SELECTIVE INHIBITORS OF Alpha2-CONTAINING ISOFORMS OF Na,K-ATPase AND USE THEREOF FOR REDUCTION OF INTRAOCULAR PRESSURE

ABSTRACT

Provided herein are alpha2-selective Na,K-ATPase inhibitors and prodrugs thereof, characterized by having a cyclic moiety attached to a digoxin or digitoxin derivative, as well as uses thereof in lowering intraocular pressure and in treating glaucoma and heart conditions.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/745,441 filed on Jan. 17, 2018, which is a National Phase of PCT Patent Application No. PCT/IL2016/050785 having International Filing Date of Jul. 19, 2016, which claims the benefit of priority under 35 USC § 119(e) of U.S. Provisional Patent Application No. 62/302,226, filed on Mar. 2, 2016. PCT Patent Application No. PCT/IL2016/050785 is also a Continuation-in-Part (CIP) of PCT Patent Application No. PCT/IL2015/050741, having international Filing Date of Jul. 19, 2015. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 82372SequenceListing.txt, created on Apr. 13, 2020, comprising 73,501 bytes, submitted concurrently with the filing of this application is incorporated herein by reference. The sequence listing submitted herewith is identical to the sequence listing forming part of the international application.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to pharmaceutical agents and, more particularly, but not exclusively, to digoxin and digitoxin derivatives exhibiting selective inhibition of α2-containing isoforms of Na,K-ATPase, and uses thereof to reduce intraocular pressure (IOP), and/or as cardiotonic agents in a subject in need thereof.

Glaucoma is a disease leading to irreversible blindness. Control of intraocular pressure (IOP) is the mainstay of current therapy of glaucoma, and is achieved by various drugs, such as β-blockers, prostaglandin analogues, α2 adrenergic receptor agonists, cholinergic agonists and carbonic anhydrase inhibitors given topically or systemically. The topical route is preferable, provided the drug effectively permeates the cornea, because this minimizes systemic side-effects. Despite the selection of drugs available, uncontrolled IOP in many patients eventually makes surgical intervention necessary. Thus, fresh approaches to drug treatment of glaucoma are highly desirable.

The Na,K-ATPase is the motor for production of the aqueous humour (bodily fluid) in the ciliary body epithelium and, in principle, inhibition of the Na,K-ATPase can suppress the production of aqueous humour, and control IOP. Control of IOP is the mainstay of glaucoma therapy; however, the available drugs suffer from a variety of shortcomings, particularly due to systemic adverse effects and low therapeutic index. Previously, intra-venous digoxin, a classical inhibitor of the Na,K-pump, typically used primarily to treat congestive heart failure, was considered for this role but was discarded due to systemic toxicity.

Isoforms of the Na,K-ATPase ion pump consists of α and β subunits (α/β) and accessory FXYD regulatory subunits. There are four isoforms of the α1 subunit (α1-4) and three isoforms of the β subunit (β1-3) expressed in a tissue-specific fashion. The α1 isoform is the common isoform that maintains Na and K gradients in all tissues, α2 is expressed mainly in muscle and astrocytes, and α3 is expressed mainly in nerve cells. For example, human heart expresses α1 (about 70%) and both α2 and α3 isoforms (about 30%) and β1.

The ciliary epithelium in the eye is a functional syncytium consisting of apical pigmented cells (PE) oriented towards the blood and baso-lateral non-pigmented (NPE) cells oriented towards the anterior chamber of the eye. It is known that the primary Na,K-ATPase isoform of the PE is α1β1 while that of the NPE is α2β3. The Na,K-ATPase in the NPE powers the production of the aqueous humor and controls intraocular pressure.

Thus, in principle, topically applied α2-selective cardiac glycosides that penetrate the intact eye and reach the ciliary epithelium could effectively reduce IOP. A potential advantage of topical application could be that systemic toxic effects typical of cardiac glycosides should be minimal.

Another possible application of an α2-selective cardiac glycoside could be as an effective cardiotonic drug, with reduced cardiotoxicity, compared to known drugs such as digoxin. Digitalis drugs such as digoxin have been used to treat heart failure for over two hundred years but are dangerous drugs with multiple side effects. There is now good evidence that selective inhibition of α2 is especially effective in enhancing cardiac excitation-contraction coupling and mediating cardiac glycoside-mediated positive inotropy. Inhibition of α2, which is a minor isoform and is located largely in T-tubules, may mediate the positive cardiotonic effects, but α2-selective cardiac glycosides should only minimally inhibit α1, located primarily in the outer sarcolemma membrane, and thus avoid cellular Ca overload, the hallmark of cardiac toxicity.

The isoform selectivity of a large number of known cardiac glycosides has been previously studied, using the yeast P. pastoris expressing Na,K-ATPase isoforms (α1β1, α2β1, β3β1), and purified detergent-soluble isoform complexes of Na,K-ATPase [Cohen E. et al., 2005, J Biol Chem, 280(17), pp. 16610-16618; Haviv H, et al., 2007, Biochemistry, 46(44), pp. 12855-12867; Lifshitz Y, et al., 2007, Biochemistry, 46(51), pp. 14937-14950; Mishra N K, et al., 2011, J Biol Chem, 286(11), pp. 9699-9712; and Kapri-Pardes E, et al., 2011, J Biol Chem, 286(50), pp. 42888-42899].

Dissociation constants, K_(D), for digitalis glycosides, digoxin and digitoxin, measured by ³H-ouabain displacement assays in membranes, showed moderate selectivity (3-4-fold) for α2/α3 over α1. By contrast to the digitalis glycosides, the K_(D) of ouabain showed some preference for α1 over α2 and similar Ki values for all three isoforms. In assays of inhibition of Na,K-ATPase activity, measured with the purified isoform protein complexes, digoxin and digitoxin showed 3-4-fold lower Ki (inhibition) values for α2 compared to α1, with α3 more similar to α1. No aglycones of any cardiac glycosides tested showed isoform selectivity. For digoxin derivatives, with one to four digitoxose moieties the maximal α2/α1 selectivity was found for digoxin itself, with three digitoxose sugars [Katz, A. et al., J Biol Chem, 2010, 285(25), pp. 19582-19592].

Based on recent studies [Laursen, M. et al., Proc Natl Acad Sci USA, 2015, 112(6):1755-60], it was inferred that the sugar moiety of digoxin likely determines isoform selectivity, which is generally consistent with recent structures of Na,K-ATPase with bound ouabain, bufalin or digoxin. The unsaturated lactone ring and steroid portion of ouabain are bound between trans-membrane segments M1, M4, M5 of the c subunit, in which there are no amino-acid differences between isoforms. Assuming that the aglycones of all cardiac glycosides bind similarly, the implication is that isoforms cannot discriminate between any of the aglycones, as found experimentally. By contrast, the sugar is bound near extracellular loops, where there are a number of amino-acid differences between the isoforms. These residues might interact with the sugars of bound digoxin in an isoform-selective way.

Additional background art include WO 2015/029035, WO 2007/079128, WO 2010/053771, U.S. Patent Application No. 2005/0032138 and U.S. Pat. Nos. 7,888,059 and 7,851,145; these documents are hereby incorporated by reference.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a compound represented by general Formula I:

including any pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer and diastereomer thereof, and any mixtures thereof,

wherein:

X is H or OH;

R is represented by general Formula II:

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety;

or

B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide;

or

B is —NR₁R₂, wherein R₁ and R₂ are each independently H or a C₁-C₄ alkyl provided that at least one of R₁ and R₂ is a C₁-C₄ alkyl.

According to some embodiments of the invention, A is selected from the group consisting of a covalent bond, an unsubstituted C₁-C₆ alkyl, a substituted C₁-C₆ alkyl, an unsubstituted C₁-C₆ alkyl interrupted by one or more heteroatom and a substituted C₁-C₆ alkyl interrupted by one or more heteroatom.

According to some embodiments of the invention, B is a cyclic moiety selected from the group consisting of an unsubstituted alicyclic moiety, a substituted alicyclic moiety, an unsubstituted heterocyclic moiety, a substituted heterocyclic moiety, an unsubstituted aryl moiety, a substituted aryl moiety, an unsubstituted heteroaryl moiety and a substituted heteroaryl moiety.

According to some embodiments of the invention, B is an unsubstituted alicyclic moiety.

According to some embodiments of the invention, the unsubstituted alicyclic moiety is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

According to some embodiments of the invention, B is a substituted alicyclic moiety.

According to some embodiments of the invention, the substituted alicyclic moiety is selected from the group consisting of 2,3-dimethylcyclopropane-1-yl, 3,3-dimethylcyclobutane-1-yl, 3,4-dimethylcyclopentane-1-yl and 3,5-dimethylcyclohexane-1-yl.

According to some embodiments of the invention, B is an unsubstituted heterocyclic moiety.

According to some embodiments of the invention, the unsubstituted heterocyclic moiety is selected from the group consisting of oxiranyl, aziridinyl, oxetanyl, azetidinyl, thietanyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl and piperidinyl.

According to some embodiments of the invention, B is an unsubstituted aryl moiety.

According to some embodiments of the invention, the unsubstituted aryl moiety is selected from the group consisting of cyclopentadienyl, phenyl and naphthyl.

According to some embodiments of the invention, B is an unsubstituted heteroaryl moiety.

According to some embodiments of the invention, the unsubstituted heteroaryl moiety is imidazolyl.

According to some embodiments of the invention, the alkylsulfonyl is selected from the group consisting of methylsulfonyl, ethylsulfonyl and isopropylsulfonyl.

According to some embodiments of the invention, the arylsulfonyl is selected from the group consisting of phenylsulfonyl, benzylsulfonyl and tosyl.

According to some embodiments of the invention, the sulfonamide is selected from the group consisting of methylsulfonamide, N-methylmethanesulfonamide and N,N-dimethylmethanesulfonamide.

According to some embodiments of the invention, B is —N(Et)₂.

According to some embodiments of the invention, X is H.

According to some embodiments of the invention, A is a covalent bond and B is cyclobutyl.

According to some embodiments of the invention, A is —CH₂— and B is cyclopropyl.

According to some embodiments of the invention, X is OH.

According to some embodiments of the invention, A is a covalent bond and B is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

According to some embodiments of the invention, A is —CH₂— and B is selected from the group consisting of cyclopropyl, 3,3-dimethylcyclobutane-1-yl and phenyl.

According to some embodiments of the invention, A is —(CH₂)₂— and B is cyclopropyl.

According to some embodiments of the invention, R is selected from the group consisting of cyclopropyl, methylcyclopropane, ethylcyclopropane, propylcyclopropane, cyclobutyl, methylcyclobutane, methyl-3,3-dimethylcyclobutane, ethylcyclobutane, propylcyclobutane, cyclopentyl, methylcyclopentane, ethylcyclopentane, propylcyclopentane, cyclohexyl, azetidinyl, oxetanyl, thietanyl, histaminyl and benzyl.

According to some embodiments of the invention, R is selected from the group consisting of cyclopropyl, methylcyclopropane and cyclobutyl.

According to some of any of the embodiments of the invention, the compound is having an affinity to at least one isoform of Na,K-ATPase.

According to some embodiments of the invention, the isoform is selected from the group consisting of α1β1, α1β2, α1β3, α2β1, β2β2, β2β3, α3β1, α3β2, α3β3, α4β1, α4β2 and α4β3.

According to some embodiments of the invention, the affinity of the compound to any one of α2β1, α2β2 and α2β3 is higher than the affinity to α1β1, α1δ2, α1β3, α3β1, α3β2, α3β3, α4β1, α4β2 and α4β3 by at least 100%.

According to some embodiments of the invention, the affinity of the compound to any one of α2β1, α2β2 and α2β3 is higher than the affinity to α1β1 by at least 300% (4-fold).

According to some embodiments of the invention, the affinity of the compound to α2β3 is higher than the affinity to α1β1 by at least 500% (6-fold).

According to some embodiments of the invention, the compound is having a α2β3 inhibition constant (Ki) lower than 10 nM.

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition that includes as an active ingredient a compound according to any of the embodiments of the invention and a pharmaceutically acceptable carrier.

According to some embodiments of the invention, the pharmaceutical composition is packaged in a packaging material and identified in print, or on the packaging material, for use in reducing intraocular pressure (IOP).

According to some embodiments of the invention, the pharmaceutical composition is packaged in a packaging material and identified in print, or on the packaging material, for use in a treatment of a heart condition.

According to an aspect of some embodiments of the present invention there is provided a method of reducing intraocular pressure (IOP) in a subject in need thereof, which includes administering to the subject a therapeutically effective amount of a compound according to any of the embodiments of the invention, or a pharmaceutical composition o according to some of the embodiments of the invention.

According to an aspect of some embodiments of the present invention there is provided a method of treating a heart condition in a subject in need thereof, that includes administering to the subject a therapeutically effective amount of a compound according to any of the embodiments of the invention, or a pharmaceutical composition according to some of the embodiments of the invention.

According to an aspect of some embodiments of the present invention there is provided a use of a compound according to any of the embodiments of the invention, or a pharmaceutical composition according to some of the embodiments of the invention, for the manufacture of a medicament for reducing intraocular pressure (IOP).

According to an aspect of some embodiments of the present invention there is provided a use of a compound according to any of the embodiments of the invention, or a pharmaceutical composition according to some of the embodiments of the invention, for the manufacture of a medicament for treating a heart condition.

According to some embodiments of the invention, the heart condition is selected from the group consisting of atrial fibrillation, atrial flutter, mitral stenosis, chronic heart failure and congestive heart failure.

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition that includes as active ingredients:

at least one ingredient selected from the group consisting of a prostaglandin analog, a β-blocker, an adrenergic agent, an α2-adrenergic receptor agonist, a miotic agent, a carbonic anhydrase inhibitor and a cholinergic agonist; and

a compound represented by Formula III:

including any pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer and diastereomer thereof, and any mixtures thereof, and a pharmaceutically acceptable carrier,

wherein:

X is H or OH;

R′ is selected from the group consisting of OH, C₁-C₆ alkyl, C₁-C₆ haloalkyl (C₁-C₆ alkyl substituted with at least one halo), —(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂, —(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂ and —(CR^(b)R^(c))n-NH₂;

Y is O or S;

R1, R2 and R3 are each independently H or a C₁-C₄ alkyl;

Ra is a C₁-C₄ alkyl;

Rb, Rc and Rd are each independently selected from H, a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl;

Re is selected from a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; and

n is 0, 1 or 2;

or R′ is represented by general Formula II:

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety, or B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is —NR₁R₂, wherein R₁ and R₂ are each independently H or a C₁-C₄ alkyl provided that at least one of R₁ and R₂ is a C₁-C₄ alkyl,

and a pharmaceutically acceptable carrier.

According to some embodiments of the invention, the pharmaceutical composition is packaged in a packaging material and identified in print, or on the packaging material, for use in reducing intraocular pressure (IOP).

According to an aspect of some embodiments of the present invention there is provided a use of an agent selected from the group consisting of a prostaglandin analog, a β-blocker, an adrenergic agent, an α2-adrenergic receptor agonist, a miotic agent, a carbonic anhydrase inhibitor and a cholinergic agonist, and a compound represented by Formula III:

including any pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer and diastereomer thereof, and any mixtures thereof,

wherein:

X is H or OH;

R′ is selected from the group consisting of OH, C₁-C₆ alkyl, C₁-C₆ haloalkyl (C₁-C₆ alkyl substituted with at least one halo), —(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂, —(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂ and —(CR^(b)R^(c))n-NH₂;

Y is O or S;

R1, R2 and R3 are each independently H or a C₁-C₄ alkyl;

Ra is a C₁-C₄ alkyl;

Rb, Rc and Rd are each independently selected from H, a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl;

Re is selected from a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; and

n is 0, 1 or 2;

or R′ is represented by general Formula II:

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety, or B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is —NR₁R₂, wherein R₁ and R₂ are each independently H or a C₁-C₄ alkyl provided that at least one of R₁ and R₂ is a C₁-C₄ alkyl,

for the manufacture of a medicament for reducing intraocular pressure (IOP).

According to an aspect of some embodiments of the present invention there is provided a method of reducing intraocular pressure (IOP) in a subject in need thereof, which includes co-administering to the subject a therapeutically effective amount of:

an agent selected from the group consisting of a prostaglandin analog, a β-blocker, an adrenergic agent, an α2-adrenergic receptor agonist, a miotic agent, a carbonic anhydrase inhibitor and a cholinergic agonist; and

a compound represented by Formula III:

including any pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer and diastereomer thereof, and any mixtures thereof,

wherein:

X is H or OH;

R′ is selected from the group consisting of OH, C₁-C₆ alkyl, C₁-C₆ haloalkyl (C₁-C₆ alkyl substituted with at least one halo), —(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂, —(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂ and —(CR^(b)R^(c))n-NH₂;

Y is O or S;

R₁, R₂ and R₃ are each independently H or a C₁-C₄ alkyl;

Ra is a C₁-C₄ alkyl;

Rb, Rc and Rd are each independently selected from H, a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl;

Re is selected from a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; and

n is 0, 1 or 2;

or R′ is represented by general Formula II:

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety, or B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is —NR₁R₂, wherein R₁ and R₂ are each independently H or a C₁-C₄ alkyl provided that at least one of R₁ and R₂ is a C₁-C₄ alkyl.

According to some embodiments of the invention, the mode of administration is effected topically, extraocularly, intraocularly and/or intravitreally.

According to some embodiments of the invention, the composition according to some embodiments of the invention is formulated as an ophthalmic composition suitable for topical, extraocular, intraocular and/or intravitreal administration to the eye of the subject.

According to some embodiments of the invention, the composition according to some embodiments of the invention is in the form selected from the group consisting of an eye-drop solution, a spray, an eye wash solution, an ointment, a suspension, a gel, a cream and an injectable solution.

According to some embodiments of the invention, the intraocular pressure (IOP) is associated with glaucoma, low-tension glaucoma and normal-tension glaucoma.

According to an aspect of some embodiments of the present invention there is provided a method of treating a heart condition in a subject in need thereof, that includes co-administering to the subject a therapeutically effective amount of:

an agent selected from the group consisting of a β-blocker, an anticoagulation agent, an angiotensin-converting-enzyme inhibitor and an angiotensin II receptor antagonist; and

a compound represented by Formula III:

including any pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer and diastereomer thereof, and any mixtures thereof,

wherein:

X is H or OH;

R′ is selected from the group consisting of OH, C₁-C₆ alkyl, C₁-C₆ haloalkyl (C₁-C₆ alkyl substituted with at least one halo), —(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂, —(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂ and —(CR^(b)R^(c))n-NH₂;

Y is O or S;

R₁, R₂ and R₃ are each independently H or a C₁-C₄ alkyl;

Ra is a C₁-C₄ alkyl;

Rb, Rc and Rd are each independently selected from H, a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl;

Re is selected from a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; and

n is 0, 1 or 2;

or R′ is represented by general Formula II:

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety, or B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is —NR₁R₂, wherein R₁ and R₂ are each independently H or a C₁-C₄ alkyl provided that at least one of R₁ and R₂ is a C₁-C₄ alkyl.

According to an aspect of some embodiments of the present invention there is provided a process of preparing a compound according any of the embodiments of the invention, the process includes:

converting the third digitoxose moiety of digoxin or digitoxin into a dialdehyde; and

reacting the dialdehyde with a reagent represented by general formula IV:

B-A-NH₂   Formula IV

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety, or B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is —NR₁R₂, wherein R₁ and R₂ are each independently H or a C₁-C₄ alkyl provided that at least one of R₁ and R₂ is a C₁-C₄ alkyl.

According to some embodiments of the invention, converting the third digitoxose moiety of digoxin or digitoxin into a dialdehyde is effected by sodium periodate (NaIO₄).

According to some embodiments of the invention, reacting the dialdehyde with a reagent represented by general formula IV is effected in the presence of NaCNBH₃.

According to an aspect of some embodiments of the present invention there is provided a method of determining an affinity of a compound, according to any embodiment of the invention, to at least one isoform of Na,K-ATPase, the method includes contacting the isoform of Na,K-ATPase with the compound in an affinity measurement setup and determining the affinity.

According to an aspect of some embodiments of the present invention there is provided a method of isolating an isoform of Na,K-ATPase of a mammal, that includes:

transforming yeast cells with a clone that that includes an α chain sequence and a β chain sequence of the Na,K-ATPase;

expressing the clone in the yeast cells; and

isolating the isoform,

wherein:

the α chain sequence is selected from the group consisting of α1, α2, α3 and α4; and

the β chain sequence is selected from the group consisting of β1, β2 and β3.

According to some embodiments of the invention, the isoform is selected from the group consisting of α1β1, β1β2, α1β3, α2β1, α2β2, α2β3, α3β1, α3β2, α3β3, α4β1, α4β2 and α4β3.

According to some embodiments of the invention, the isoform is α2β2.

According to some embodiments of the invention, the isoform is α2β3.

According to an aspect of some embodiments of the present invention there is provided an isolated isoform of Na,K-ATPase of a mammal having at least 70% purity, wherein the isoform is α2β2.

According to an aspect of some embodiments of the present invention there is provided an isolated isoform of Na,K-ATPase of a mammal having at least 70% purity, wherein the isoform is α2β3.

According to an aspect of some embodiments of the present invention there is provided an isolated isoform of Na,K-ATPase of a mammal having a yeast-characterizing glycosylation pattern, wherein the isoform is α2β2.

According to an aspect of some embodiments of the present invention there is provided an isolated isoform of Na,K-ATPase of a mammal having a yeast-characterizing glycosylation pattern, wherein the isoform is α2β3.

According to some embodiments of the invention, the isolated isoform, according to any embodiment of the invention, is human.

According to some embodiments of the invention a prodrug of the compound is represented by Formula V:

wherein X is H or PD₃, and

each of PD₁-PD₄ is H or independently selected from the group consisting of a methoxymethyl ether, a tetrahydropyranyl ether, a t-butyl ether, an allyl ether, a benzyl ether, a t-butyldimethylsilyl ether, a t-butyldiphenylsilyl ether, an acetic acid ester (Ac), ethyl, propyl, butyl, t-butyl or pivalic acid ester and a benzoic acid ester, provided that at least one of PD₁-PD₄ is not H.

According to some embodiments, each of PD₁-PD₄ is an acetic acid ester (Ac).

According to some embodiments, each of PD₁ and PD₂ is an acetic acid ester (Ac).

According to some embodiments, each of PD₁-PD₃ is an acetic acid ester (Ac).

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A, 1B, 1C and 1D present comparative plots of IOP as a function of time, showing the dose response of α2-inhibitor compounds, according to some embodiments of the present invention, in lowering IOP in live rabbits, wherein FIG. 1A shows the results obtained for DiB, FIG. 1B shows the duration of the effect of DiB while 4AP is added every 2 hours so as to maintain the raised IOP, FIG. 1C shows the results obtained for DMcP, and FIG. 1D shows the results obtained for DcB;

FIGS. 2A, 2B, 2C and 2D present comparative plots of IOP as a function of time, demonstrating the capacity of the α2-inhibitor compounds, according to some embodiments of the present invention, to lower IOP below basal levels compared to a buffer control when administered topically to one eye of a rabbit, while the other eye received PBS as a control, wherein FIG. 2A shows the lack of effect of digoxin, FIG. 2B shows the lack of effect of DiB (a non-cyclic moiety inhibitor), FIG. 2C shows the notable effect of DMcP, and FIG. 2D shows the notable effect of DcB;

FIGS. 3A, 3B and 3C present comparative plots of IOP as a function of time, demonstrating the effect of α2-inhibitor compounds, according to some embodiments of the present invention, to potentiate the drug Latanoprost in lowering IOP below basal levels, wherein FIG. 3A shows the effect of DcB alone, FIG. 3B shows the effect of Latanoprost alone, and FIG. 3C shows the effect of co-administering DcB with Latanoprost; and

FIG. 4 presents comparative plots of IOP as a function of time, demonstrating the capacity of the trisAcDcB prodrug of the α2-inhibitor compound DcB, according to some embodiments of the present invention, to lower IOP below basal levels compared to a buffer control when administered topically to one eye of a rabbit, while the other eye received PBS as a control.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a pharmaceutical agents and, more particularly, but not exclusively, to digoxin and digitoxin derivatives exhibiting selective inhibition of α2-containing isoforms of Na,K-ATPase, and uses thereof to reduce intraocular pressure (IOP), and/or as cardiotonic agents in a subject in need thereof.

The principles and operation of the present invention may be better understood with reference to the figures and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

As discussed hereinabove, Na,K-ATPase inhibitors that exhibit selectivity towards tissue-specific isoforms of the protein, present pharmaceutical advantages such as broader therapeutic window and wider scope of modes of administration. For example, Na,K-ATPase inhibitors exhibiting selectivity towards protein isoforms containing the α2 subunit offer this advantages over unselective inhibitors in treating medical conditions wherein lowering the intraocular pressure is called for.

While searching for α2-selective inhibitors, the present inventors have surprisingly found that certain derivatives of digoxin and digitoxin, wherein the perhydro-1-4-oxazepine moiety thereof is N-substituted with a cyclic moiety, exhibit a notable selectivity towards α2-containing isoform of Na,K-ATPase.

Compounds:

According to an aspect of some embodiments of the present invention there is provided a compound represented by general Formula I:

wherein:

X is H or OH, whereas derivatives having ×=H are referred to as digitoxin derivatives, and derivatives having X=—OH are referred to as digoxin derivatives;

R is represented by general Formula II:

the wiggled line represents the N-link to the compound;

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety;

or

R is represented by general Formula II:

the wiggled line represents the N-link to the compound;

A is a spacer moiety or a covalent bond; and

B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide;

or

R is represented by general Formula II:

the wiggled line represents the N-link to the compound;

A is a spacer moiety or a covalent bond; and

B is —NR₁R₂, wherein R₁ and R₂ are each independently H or a C₁-C₄ alkyl provided that at least one of R₁ and R₂ is a C₁-C₄ alkyl, namely B is a secondary amine or tertiary amine.

As used herein, the term “cyclic moiety” refers to a group of atoms that are covalently attached to one another so as to form at least one ring of atoms. Non-limiting examples of cyclic moieties include unsubstituted alicyclic moieties, substituted alicyclic moieties, unsubstituted heterocyclic moieties, substituted heterocyclic moieties, unsubstituted aryl moieties, substituted aryl moieties, unsubstituted heteroaryl moiety and substituted heteroaryl moieties.

A substituted cyclic moiety has one or more chemical group or atom attached to one of the atoms in the ring of atoms. Examples of such chemical groups or atoms include, without limitation, C₁-C₆ alkyl, hydroxyl, amine, halo, alkoxy, carboxyl, amide and the like, or a second cyclic moiety attached by a covalent bond(s) to one or two of the ring atoms of the cyclic moiety.

The terms “hydroxyl” or “hydroxy”, as used herein, refer to an —OH group.

As used herein, the term “amine” describes a —NR¹R² group where each of R¹ and R² is independently hydrogen, alkyl, cycloalkyl, heteroalicyclic, aryl or heteroaryl, as these terms are defined herein.

As used herein, the term “alkyl” describes an aliphatic hydrocarbon including straight chain and branched chain groups. The alkyl may have 1 to 20 carbon atoms, or 1-10 carbon atoms, and may be branched or unbranched. According to some embodiments of the present invention, the alkyl is a low (or lower) alkyl, having 1-4 carbon atoms (namely, methyl, ethyl, propyl and butyl).

Whenever a numerical range; e.g., “1-10”, is stated herein, it implies that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. In some embodiments, the alkyl is a lower alkyl, including 1-6 or 1-4 carbon atoms.

A C₁-C₆ alkyl group refers to any one of the moieties methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, t-pentyl, neopentyl, i-pentyl, s-pentyl, 3-pentyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl and 2,3-dimethylbutyl.

The term “halide”, as used herein, refers to the anion of a halo atom, i.e. F⁻, Cl⁻, Br⁻ and I⁻.

The term “halo” refers to F, Cl, Br and I atoms as substituents.

The term “alkoxy” refers to an —OR¹ group, wherein R¹ is as defined herein, but other than hydrogen.

The term “amide” as used herein encompasses C-amide and N-amide.

The term “C-amide” describes a —C(═O)—NR¹R² group, where R¹ and R² are as defined herein.

The term “N-amide” describes a R¹C(═O)—NR²— group, where R¹ and R² are as defined herein.

The term alkylsulfonyl and arylsulfonyl refers to an R¹—S(═O)₂— group, wherein R¹ is as defined herein, but other than hydrogen. Examples of arylsulfonyl groups include p-toluenesulfonyl (tosyl; Ts), p-bromobenzenesulfonyl (brosyl; Bs), 2- or 4-nitrobenzenesulfonyl (nosyl; Ns), methanesulfonyl (mesyl; Ms), trifluoromethanesulfonyl (triflyl; Tf), and 5-(dimethylamino)naphthalene-1-sulfonyl (Dansyl; Ds).

The term solnfonamide refers to an R³—S(═O)₂— group, wherein R³ is amine as defined herein.

The terms “alicyclic” and “cycloalkyl”, refer to an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms), branched or unbranched group containing 3 or more carbon atoms where one or more of the rings does not have a completely conjugated pi-electron system, and may further be substituted or unsubstituted. The cycloalkyl can be substituted or unsubstituted.

Examples of alicyclic moieties include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclododecyl.

Examples of substituted alicyclic is selected from the group consisting of 2,3-dimethylcyclopropane-1-yl, 3,3-dimethylcyclobutane-1-yl, 3,4-dimethylcyclopentane-1-yl and 3,5-dimethylcyclohexane-1-yl.

The terms “heterocyclic” or “heteroalicyclic”, as used herein, describe a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. The heteroalicyclic may be substituted or unsubstituted. Representative examples are morpholine, piperidine, piperazine, tetrahydrofurane, tetrahydropyrane and the like.

In some embodiments, the heterocyclic moiety is selected from the group consisting of oxiranyl, aziridinyl, oxetanyl, azetidinyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl and piperidinyl.

The term “aryl” describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system, as in the example of phenyl. The aryl group may be unsubstituted or substituted by one or more substituents. Examples of aryls include cyclopentadienyl, phenyl and naphthyl.

The term “heteroaryl” describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl moieties include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine, thiadiazole, indole and the like. The heteroaryl group may be unsubstituted or substituted by one or more substituents.

According to some embodiments, the spacer moiety A can be a covalent bond, an unsubstituted C₁-C₆ alkyl, a substituted C₁-C₆ alkyl, an unsubstituted C₁-C₆ alkyl interrupted by one or more heteroatom (e.g., O, N or S) and a substituted C₁-C₆ alkyl interrupted by one or more heteroatom. A spacer moiety may be substituted with one or more C₁-C₆ alkyl, hydroxyl, amine, halo, alkoxy, carboxyl, amide and the like.

In some embodiments, A is a covalent bond and B an alicyclic moiety such as, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the corresponding R in Formula I is:

In some embodiments, A is a covalent bond and B a heteroalicyclic moiety such as, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the corresponding R in Formula I is:

In some embodiments, A is —CH₂— and B a cyclic moiety such as, for example, cyclopropyl, 3,3-dimethylcyclobutane-1-yl and phenyl, and the corresponding R in Formula I is:

In some embodiments, A is —(CH₂)₂— and B a cyclic moiety such as, for example, cyclopropyl, and the corresponding R in Formula I is:

In some embodiments, A is —(CH₂)₂— and B a heteroaryl cyclic moiety such as, for example, imidazolyl, and the corresponding R in Formula I is:

In some embodiment, X is H, A is a covalent bond and B is cyclobutyl.

In some embodiment, X is H, A is —CH₂— and B is cyclopropyl.

In some embodiment, X is OH, A is a covalent bond and B is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

In some embodiment, X is OH, A is —CH₂— and B is cyclopropyl, 3,3-dimethylcyclobutane-1-yl and phenyl.

In some embodiment, X is OH, A is —(CH₂)₂— and B is cyclopropyl.

In some embodiments, R of general Formula I is cyclopropyl, methylcyclopropane, ethylcyclopropane, propylcyclopropane, cyclobutyl, methylcyclobutane, methyl-3,3-dimethylcyclobutane, ethylcyclobutane, propylcyclobutane, cyclopentyl, methylcyclopentane, ethylcyclopentane, propylcyclopentane, cyclohexyl and benzyl. Alternatively, R is cyclopropyl, methylcyclopropane and cyclobutyl.

In some embodiments, A is —CH₂— and B an alkylsulfonyl such as, for example, methylsulfonyl, and the corresponding R in Formula I is:

In some embodiments, A is —(CH₂)₂— and B an alkylsulfonyl such as, for example, methylsulfonyl, or a solnfonamide, and the corresponding R in Formula I is:

In some embodiments, A is —(CH₂)₂— and B a tertiary amine such as, for example, N,N-dimethylamine, and the corresponding R in Formula I is:

In some embodiments, the compound is any one of the structures presented below:

DcP or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2R,4S,5S,6R)-5-(((2S,4S,5S,6R)-5-((4-cyclopropyl-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DMcP or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2R,4S,5S,6R)-5-(((2S,4S,5S,6R)-5-((4-(cyclopropylmethyl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DEcP or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5S,6R)-5-((4-(2-cyclopropylethyl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DcB or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2R,4S,5S,6R)-5-(((2S,4S,5S,6R)-5-((4-cyclobutyl-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DcPe or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5S,6R)-5-((4-cyclopentyl-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DcHe or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5S,6R)-5-((4-cyclohexyl-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DBz or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5S,6R)-5-((4-benzyl-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DMDMcB or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5 S,6R)-5-((4-((3,3-dimethylcyclobutyl)methyl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DtxMcP or 4-((3S,5R,8R,9S,10S,13R,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5S,6R)-5-((4-(cyclopropylmethyl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-14-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DtxcB or 4-((3S,5R,8R,9S,10S,13R,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5S,6R)-5-((4-cyclobutyl-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-14-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DAz or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2R,4S,5S,6R)-5-(((2S,4S,5S,6R)-5-((4-(azetidin-3-yl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DOx or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12,14-dihydroxy-3-(((2R,4S,5S,6R)-4-hydroxy-5-(((2S,4S,5S,6R)-4-hydroxy-6-methyl-5-((2-methyl-4-(oxetan-3-yl)-1,4-oxazepan-7-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DTh or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12,14-dihydroxy-3-(((2R,4S,5S,6R)-hydroxy-5-(((2S,4S,5S,6R)-4-hydroxy-6-methyl-5-((2-methyl-4-(thietan-3-yl)-1,4-oxazepan-7-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one; and

DHis or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2R,4S,5S,6R)-5-(((2S,4S,5S,6R)-5-((4-(2-(1H-imidazol-5-yl)ethyl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

In some embodiments, the compound is any one of the structures presented below:

DMSM or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12,14-dihydroxy-3-(((2S,4S,5R,6R)-4-hydroxy-5-(((2S,4S,5S,6R)-4-hydroxy-6-methyl-5-((2-methyl-4-((methylsulfonyl)methyl)-1,4-oxazepan-7-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DESM or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12,14-dihydroxy-3-(((2S,4S,5R,6R)-4-hydroxy-5-(((2S,4S,5S,6R)-4-hydroxy-6-methyl-5-((2-methyl-4-(2-(methylsulfonyl)ethyl)-1,4-oxazepan-7-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one; and

DESA or 2-(7-(((2R,3S,4S,6S)-6-(((2R,3R,4S,6S)-6-(((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-hydroxy-2-methyltetrahydro-2H-pyran-3-yl)oxy)-4-hydroxy-2-methyltetrahydro-2H-pyran-3-yl)oxy)-2-methyl-1,4-oxazepan-4-yl)ethanesulfonamide

In some embodiments, the compound is any one of the structures presented below:

DEDA or 4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2R,4S,5S,6R)-5-(((2S,4S,5S,6R)-5-((4-(2-(dimethylamino)ethyl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

The present embodiments further encompass any enantiomers, diastereomers, optical isomers, prodrugs, solvates, hydrates, polymorphs, geometrical isomers and/or pharmaceutically acceptable salts of the compounds described herein.

Any one or more of the compounds presented herein may be present as a salt. The term “salt” encompasses both basic and acid addition salts, and include salts formed with organic and inorganic anions and cations. The term “organic or inorganic cation” refers to counter-ions for an acid. The counter-ions can be chosen from the alkali and alkaline earth metals, (such as lithium, sodium, potassium, barium, aluminum and calcium), ammonium and the like. Furthermore, the term includes salts that form by standard acid-base reactions of basic groups and organic or inorganic acids. Such acids include hydrochloric, hydrofluoric, hydrobromic, trifluoroacetic, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, cholic, pamoic, mucic, D-camphoric, phthalic, tartaric, salicylic, methanesulfonic, benzenesulfonic, p-toluenesulfonic, sorbic, picric, benzoic, cinnamic, and like acids.

As used herein, the phrase “pharmaceutically acceptable salt” refers to a charged species of the parent compound and its counter-ion, which is typically used to modify the solubility characteristics of the parent compound and/or to reduce any significant irritation to an organism by the parent compound, while not abrogating the biological activity and properties of the administered compound. A pharmaceutically acceptable salt of a compound as described herein can alternatively be formed during the synthesis of the compound, e.g., in the course of isolating the compound from a reaction mixture or re-crystallizing the compound.

In the context of some of the present embodiments, a pharmaceutically acceptable salt of the compounds described herein may optionally be an acid addition salt comprising at least one basic (e.g., amine and/or guanidine) group of the compound which is in a positively charged form (e.g., wherein the basic group is protonated), in combination with at least one counter-ion, derived from the selected base, that forms a pharmaceutically acceptable salt.

The acid addition salts of the compounds described herein may therefore be complexes formed between one or more basic groups of the compound and one or more equivalents of an acid.

Depending on the stoichiometric proportions between the charged group(s) in the compound and the counter-ion in the salt, the acid additions salts can be either mono-addition salts or poly-addition salts.

The phrase “mono-addition salt”, as used herein, refers to a salt in which the stoichiometric ratio between the counter-ion and charged form of the compound is 1:1, such that the addition salt includes one molar equivalent of the counter-ion per one molar equivalent of the compound.

The phrase “poly-addition salt”, as used herein, refers to a salt in which the stoichiometric ratio between the counter-ion and the charged form of the compound is greater than 1:1 and is, for example, 2:1, 3:1, 4:1 and so on, such that the addition salt includes two or more molar equivalents of the counter-ion per one molar equivalent of the compound.

An example, without limitation, of a pharmaceutically acceptable salt would be an ammonium cation or guanidinium cation and an acid addition salt thereof.

The acid addition salts may include a variety of organic and inorganic acids, such as, but not limited to, hydrochloric acid which affords a hydrochloric acid addition salt, hydrobromic acid which affords a hydrobromic acid addition salt, acetic acid which affords an acetic acid addition salt, ascorbic acid which affords an ascorbic acid addition salt, benzenesulfonic acid which affords a besylate addition salt, camphorsulfonic acid which affords a camphorsulfonic acid addition salt, citric acid which affords a citric acid addition salt, maleic acid which affords a maleic acid addition salt, malic acid which affords a malic acid addition salt, methanesulfonic acid which affords a methanesulfonic acid (mesylate) addition salt, naphthalenesulfonic acid which affords a naphthalenesulfonic acid addition salt, oxalic acid which affords an oxalic acid addition salt, phosphoric acid which affords a phosphoric acid addition salt, toluenesulfonic acid which affords a p-toluenesulfonic acid addition salt, succinic acid which affords a succinic acid addition salt, sulfuric acid which affords a sulfuric acid addition salt, tartaric acid which affords a tartaric acid addition salt and trifluoroacetic acid which affords a trifluoroacetic acid addition salt. Each of these acid addition salts can be either a mono-addition salt or a poly-addition salt, as these terms are defined herein.

As used herein, the term “enantiomer” refers to a stereoisomer of a compound that is superposable with respect to its counterpart only by a complete inversion/reflection (mirror image) of each other. Enantiomers are said to have “handedness” since they refer to each other like the right and left hand. Enantiomers have identical chemical and physical properties except when present in an environment which by itself has handedness, such as all living systems. In the context of the present embodiments, a compound may exhibit one or more chiral centers, each of which exhibiting an R- or an S-configuration and any combination, and compounds according to some embodiments of the present invention, can have any their chiral centers exhibit an R- or an S-configuration.

The term “diastereomers”, as used herein, refers to stereoisomers that are not enantiomers to one another. Diastereomerism occurs when two or more stereoisomers of a compound have different configurations at one or more, but not all of the equivalent (related) stereocenters and are not mirror images of each other. When two diastereoisomers differ from each other at only one stereocenter they are epimers. Each stereo-center (chiral center) gives rise to two different configurations and thus to two different stereoisomers. In the context of the present invention, embodiments of the present invention encompass compounds with multiple chiral centers that occur in any combination of stereo-configuration, namely any diastereomer.

All stereoisomers, optical and geometrical isomers of the compounds of the present invention are contemplated, either in admixture or in pure or substantially pure form. The compounds of the present invention can have asymmetric centers at one or more of the atoms. Consequently, the compounds can exist in enantiomeric or diastereomeric forms or in mixtures thereof. The present invention contemplates the use of any racemates (i.e. mixtures containing equal amounts of each enantiomers), enantiomerically enriched mixtures (i.e., mixtures enriched for one enantiomer), pure enantiomers or diastereomers, or any mixtures thereof. The chiral centers can be designated as R or S or R,S or d,D, l,L or d,l, D,L.

The term “prodrug” refers to an agent, which is converted into the active compound (the active parent drug) in vivo. Prodrugs are typically useful for facilitating the administration of the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. A prodrug may also have improved solubility as compared with the parent drug in pharmaceutical compositions. Prodrugs are also often used to achieve a sustained release of the active compound in vivo. An example, without limitation, of a prodrug would be a compound of the present invention, having one or more carboxylic acid moieties, which is administered as an ester (the “prodrug”). Such a prodrug is hydrolyzed in vivo, to thereby provide the free compound (the parent drug). The selected ester may affect both the solubility characteristics and the hydrolysis rate of the prodrug.

The term “solvate” refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by a solute (the compound of the present invention) and a solvent, whereby the solvent does not interfere with the biological activity of the solute. Suitable solvents include, for example, ethanol, acetic acid and the like.

The term “hydrate” refers to a solvate, as defined hereinabove, where the solvent is water.

The present invention also includes solvates of the compounds of the present invention and salts thereof. “Solvate” means a physical association of a compound of the invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation. “Solvate” encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates and the like. “Hydrate” is a solvate wherein the solvent molecule is water.

The present invention also includes polymorphs of the compounds of the present invention and salts thereof. The term “polymorph” refers to a particular crystalline state of a substance, which can be characterized by particular physical properties such as X-ray diffraction, IR spectra, melting point, and the like.

According to some aspects of some embodiments of the present invention, the compound is represented by general Formula III:

wherein:

X is H or OH;

R′ is selected from the group consisting of OH, C₁-C₆ alkyl, C₁-C₆ haloalkyl (C₁-C₆ alkyl substituted with at least one halo), —(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂, —(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂ and —(CR^(b)R^(c))n-NH₂;

Y is O or S;

R₁, R₂ and R₃ are each independently H or a C₁-C₄ alkyl;

Ra is a C₁-C₄ alkyl;

R^(b), R^(c), R^(d) and R^(e) are each independently selected from H, a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; and

n is 0, 1 or 2;

or

R′ is represented by general Formula II:

the wiggled line represents the N-link to the compound;

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety;

or

R′ is represented by general Formula II:

the wiggled line represents the N-link to the compound;

A is a spacer moiety or a covalent bond; and

B is selected from the group consisting of an alkylsulfonyl, an arylsulfonyl and a sulfonamide;

or

R′ is represented by general Formula II:

the wiggled line represents the N-link to the compound;

A is a spacer moiety or a covalent bond; and

B is —NR₁R₂, wherein R₁ and R₂ are each independently H or a C₁-C₄ alkyl provided that at least one of R₁ and R₂ is a C₁-C₄ alkyl, including any pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer and diastereomer thereof, and any mixtures thereof, and a pharmaceutically acceptable carrier.

Additional alternative structures of R′ are described in WO 2015/029035, which is hereby specifically incorporated by reference as if fully set forth herein, thereby describing each and all such additional alternative structures.

Process of Preparing the Compounds:

According to an aspect of some embodiments of the present invention, there is provided a process for preparing the compounds represented by general Formula I, the process includes:

converting the third digitoxose moiety of digoxin or digitoxin into a dialdehyde; and

reacting said dialdehyde with a reagent represented by general formula IV:

B-A-NH₂   Formula IV

A is a spacer moiety or a covalent bond as described hereinabove; and

B is selected form the group consisting of a cyclic moiety, an alkylsulfonyl, arylsulfonyl, a sulfonamide, a secondary amine and a tertiary amine, as described hereinabove.

As can be seen in Scheme 1, the conversion of the third digitoxose moiety of digoxin or digitoxin is typically effected by selective oxidation thereof into a dialdehyde by reacting digoxin or digitoxin with a reagent that breaks apart 1,2-diols (vicinal diols) to afford aldehydes and/or ketones, such as sodium periodate (NaIO₄); The reaction of the dialdehyde is effected by reductive amination of the dialdehyde using a free amine of R (R—NH₂) in the presence of, e.g., NaCNBH₃.

Selective Affinity and Inhibition:

The results presents in the Examples section below (see, e.g., Table 2) show clearly that compounds encompassed by general Formula I, according to embodiments of the present invention, exhibit high selectivity for α2β3 over α1β1 compared to digoxin and digitoxin. The results obtained for cyclic R-substituents (R comprising a cyclic moiety), some with 4 carbon atoms, provide a clear indication for selective structural interactions of the modified sugar of digoxin with the 33 subunit. This effect may also be combined with an enhanced interaction with the α2 subunit. The detailed enzymological study, presented hereinbelow, has been obtained with the purified detergent-soluble human Na,K-ATPase isolated isoform complexes, as well as with intact bovine ciliary NPE cells, i.e., selective inhibition of human Na,KATPase isolated isoform complexes provided corroborating indication of inhibition by the compounds provided herein also of isoform mixture (α1β1 plus α2β3) as these are expressed in native NPE cell membranes.

According to some embodiments, the compounds presented herein, such as a compound represented by general Formula I or by general Formula III, exhibit an affinity to at least one isoform of Na,K-ATPase.

In the context of embodiments of the present invention, isoforms of Na,K-ATPase include any combination of α1, α2, α3 and α4 complexed with β1, β2 and β3; hence, isoforms of Na,K-ATPase include α1β1, α1β2, α1β3, α2β1, α2β2, α2β3, α3β1, α3β2, α3β3, α4β1, α4β2 and α4β3.

According to some embodiments of the present invention, a compound represented by general Formula I or by general Formula III, has a higher affinity to isoforms containing an α2 subunit, compared to its affinity to isoforms containing of the a subunits, such as α1, as demonstrated in the Examples section that follows below.

The phrase “higher affinity to”, as used herein, is a relative term that means that a given ligand molecule (compound, inhibitor, drug, etc.) is attracted and can bind to and (form a) complex with a target entity (protein, enzyme, drug-target) more strongly compared to its binding to another target entity. Affinity can be measured directly and indirectly by a number of methodologies. In some embodiments of the present invention, the affinity is referred to in terms of Ki (inhibition constant) or K_(D) (dissociation constant), as these terms are known in the art. For example, a small value for Ki means that an inhibitor has a higher effective affinity to the enzyme relative to an affinity of the same inhibitor to another enzyme or another inhibitor to the same enzyme.

According to some embodiments of the present invention, a compound represented by general Formula I or by general Formula III, is a selective inhibitor of α2-containing isoforms of Na,K-ATPase, compared to α1-containing isoforms of Na,K-ATPase, as demonstrated in the Examples section that follows below.

The terms “selective inhibitor of α2-containing isoforms of Na,K-ATPase” or “selective inhibitor of the α2 isoform of Na,K-ATPase” or “α2-selective inhibitor”, as used herein interchangeably, refer to a compound that inhibits α2-containing isoforms of Na,K-ATPase to a greater degree than the compound inhibits other isoforms of Na,K-ATPase, such as those containing α1. In some embodiments, the compounds described herein are selective for the α2β1, α2β2 and/or α2β3 isoforms of Na,K-ATPase over the α1β3 isoform thereof. In some embodiments, the selectivity of the compound for the α2-containing isoform of Na,K-ATPase (e.g., α2β1, α2β2 and/or α2β3 isoform) is at least about 4-fold (300% more selective) over other isoforms, or at least 5-fold (400%), at least 6-fold (500%), at least 8-fold (700%), at least 10-fold, at least 16-fold, at least 20-fold, at least 30-fold, or at least 50-fold greater inhibition of the α2-containing isoform of Na,K-ATPase over other isoforms of Na,K-ATPase.

Uses of α2-Selective Inhibitors:

The experimental work presented in the Examples section below demonstrates a clear correlation between increased α2β3-selectivity of the compounds presented herein and potency and duration in reducing IOP of either pharmacologically raised or basal (normal) IOP. Thus the results support a central role of α2β3 in production of aqueous humour. The mechanism of the IOP reduction has been shown to involve inhibition of active Na and K fluxes via NPE cells and reduction of inflow of aqueous humour after topical administration of compound(s) and permeation thereof via the cornea.

Without being bound by any particular theory, it is assumed that the low Ki values for inhibition of α2β3 (Ki of about 4 nM) and hydrophobic properties of the compounds according to some embodiments of the present invention, suggest that both traits contribute to the potency and long duration of action exhibited by the compounds provided herein. The finding that the compounds provided herein are effective in reducing basal (normal) IOP as low as 25-30% (see, e.g., FIGS. 2A-D and FIGS. 3A-3C), as well as the reduction of acute 4AP-induced raised IOP (see, e.g., FIGS. 1A-1D), provides an insight into the mechanism of action of the compounds.

The compounds presented herein are useful as topical opthalmological agents for treatment of glaucoma, since they exhibit at least one of efficacy and low levels of side-effects. In relations to alternative drugs, the compounds presented herein are useful as topical opthalmological agents for treatment of glaucoma, since they have been shown to exhibit at least one of improved efficacy, extended duration of desired effects and reduced side-effects, compared to currently available drugs, exemplified in the Examples section that follows below by the first-line drug latanoprost, and iopidine, used in short-term adjunctive therapy of chronic glaucoma. In general, currently available drugs include β-adrenergic antagonists and carbonic anhydrase inhibitors, that reduce the rate of aqueous humor production, or prostaglandin analogs, cholinergic agonists and sympathomimetics, that increase the rate of outflow through the trabecular meshwork and uveoscleral pathway. In this respect, the experimental results presented hereinbelow, show 25-30% higher reduction in basal IOP effected by the compounds presented herein in the rabbits, compared to Latanoprost, the current first-line drug for treatment of glaucoma.

In principle, the ability of the compounds presented herein, according to some embodiments, to reduce the basal IOP, could be relevant not only to optical hypertension and primary open angle glaucoma but also to normotensive glaucoma for which reduction of IOP below the basal level is required.

Regarding local toxicity, the rationale for making α2β3-selective inhibitors included not only the expectation of high potency but also of minimal local side-effects. In the Examples section below it is shown that corneal swelling, used to assess such adverse effect, is not observed at least over several days of topical administration of the compounds presented herein (see, e.g., Table 4). Similar evidence for safety was observed by inspection of local redness and irritation.

Concerning possible systemic toxicity of the α2β3-selective compounds presented herein, it is expected that cardiotoxicity, such as is associated with clinical use of digoxin, would be minimal, because the α2-selective compounds are likely to be intrinsically non-cardiotoxic, compared to a non-selective cardiac glycoside, and also because the pharmacokinetic concentration is likely to be low (see, e.g., inhibition results in Table 2).

The results presented hereinbelow indicate that after repeated administration over several weeks of the compounds according to embodiments of the present invention, no signed of adverse effects have been observed, indicating that compounds represented by general Formula I or III are not toxic and exhibit essentially no adverse effects.

Thus, the α2-selective inhibitors presented herein (compounds represented by general Formula I or by general Formula III) can be used to reduce intraocular pressure in a subject in need thereof, while taking advantage of the selective affinity these inhibitors exhibit towards α2-containing isoforms of Na,K-ATPase, which minimize adverse effects associated with unselective inhibition of the other isoforms.

According to an aspect of some embodiments of the present invention, there is provided a pharmaceutical composition which includes as an active ingredient any one of the compounds represented by general Formula I and a pharmaceutically acceptable carrier.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. Examples, without limitations, of carriers are: propylene glycol, saline, emulsions and mixtures of organic solvents with water, as well as solid (e.g., powdered) and gaseous carriers.

Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the compounds presented herein into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

According to some embodiments, the administration is effected topically, extraocularly, intraocularly and/or intravitreally. In some embodiments, the pharmaceutical composition is formulated as an ophthalmic composition suitable for topical, extraocular, intraocular and/or intravitreal administration to the eye of the subject. According to some embodiments, the pharmaceutical composition of the invention is an ophthalmic composition which is administered topically onto the eye of a subject for facilitating effective intraocular levels of the compound and for preventing unnecessary and unintentional levels of the compound in other tissues and/or organs. Such a non-systemic, site-specific administration reduces the side effects associated with the compounds.

In the context of some embodiments of the present invention, topical and/or extraocular administration is effected by applying the compound(s), or compositions and medicaments comprising the compound(s) to the eye or a bodily surface near the eye. According to some embodiments, the composition may take the form of an eye-drop solution, a spray, an eye wash solution, an ointment, a lotion, a suspension, a gel or a cream. The topical pharmaceutical compositions may be in the form of eye-drops to be applied by instillation into the eye or may be in the form of a viscous ointment, gel or cream to be applied by an ointment onto the ocular surface and may contain control release means for facilitating sustained release over a prolonged period of time.

In the context of some embodiments of the present invention, intraocular and/or intravitreal administration is effected by injecting the compound(s), or compositions and medicaments comprising the compound(s) into the eye or into a bodily tissue near the eye. According to some embodiments, the composition may take the form of an injectable solution.

According to some embodiments, oral or otherwise systemic administration in a dosage effective for reducing the intraocular pressure is also possible. For example, the composition may be administered by a dermal patch for extended release.

According to some embodiments, the administration is effected orally. For oral administration, the compounds presented herein can be formulated readily by combining the compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds presented herein to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the compounds presented herein may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For injection, the compounds presented herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer with or without organic solvents such as propylene glycol, polyethylene glycol.

For transmucosal administration, penetrants are used in the formulation. Such penetrants are generally known in the art.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active aminoglicoside compounds doses.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds presented herein are conveniently delivered in the form of an aerosol spray presentation (which typically includes powdered, liquefied and/or gaseous carriers) from a pressurized pack or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compounds presented herein and a suitable powder base such as, but not limited to, lactose or starch.

The compounds presented herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the compounds preparation in water-soluble form. Additionally, suspensions of the compounds presented herein may be prepared as appropriate oily injection suspensions and emulsions (e.g., water-in-oil, oil-in-water or water-in-oil in oil emulsions). Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.

Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds presented herein to allow for the preparation of highly concentrated solutions.

Alternatively, the compounds presented herein may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The compounds presented herein may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions herein described may also comprise suitable solid of gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of compounds presented herein effective to prevent, alleviate or ameliorate symptoms of the disorder, or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any compounds presented herein used in the methods of the present embodiments, the therapeutically effective amount or dose can be estimated initially from activity assays in animals. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the mutation suppression levels as determined by activity assays (e.g., the concentration of the test compounds which achieves a substantial read-through of the truncation mutation). Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the compounds presented herein can be determined by standard pharmaceutical procedures in experimental animals, e.g., by determining the EC₅₀ (the concentration of a compound where 50% of its maximal effect is observed) and the LD₅₀ (lethal dose causing death in 50% of the tested animals) for a subject compound. The data obtained from these activity assays and animal studies can be used in formulating a range of dosage for use in human.

The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provide plasma levels of the compounds presented herein which are sufficient to maintain the desired effects, termed the minimal effective concentration (MEC). The MEC will vary for each preparation, but can be estimated from in vitro data; e.g., the concentration of the compounds necessary to achieve 50-90% expression of the whole gene having a truncation mutation, i.e. read-through of the mutation codon. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. HPLC assays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using the MEC value. Preparations should be administered using a regimen, which maintains plasma levels above the MEC for 10-90% of the time, preferable between 30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the chronic condition to be treated, dosing can also be a single periodic administration of a slow release composition described hereinabove, with course of periodic treatment lasting from several days to several weeks or until sufficient amelioration is effected during the periodic treatment or substantial diminution of the disorder state is achieved for the periodic treatment.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA (the U.S. Food and Drug Administration) approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as, but not limited to a blister pack or a pressurized container (for inhalation). The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration.

Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a compound according to the present embodiments, formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition or diagnosis, as is detailed hereinabove.

In accordance with other embodiments, the compounds presented herein may be loaded into a drug-delivery device to be inserted or implanted into the eye of the subject for allowing releasing of the compound in a controlled and continuous rate, by dissolving, diffusion or leaching, thus maintaining effective therapeutic concentration over a prolonged period of time. The drug-delivery device may be for example a biocompatible thin film loaded with the active agent, inserted for example beneath the lower eyelid. In some embodiments, the drug-delivery device is a contact lens or any other ophthalmic device, as these are known in the art.

In some embodiments, the pharmaceutical composition is packaged in a packaging material and identified in print, or on the packaging material, for use in reducing intraocular pressure (IOP).

According to an aspect of some embodiments of the present invention, there is provided a use of the compounds represented by general Formula I, or a pharmaceutical composition comprising the same, for the manufacture of a medicament for reducing intraocular pressure (IOP).

According to an aspect of some embodiments of the present invention, there is provided a method of reducing intraocular pressure (IOP) in a subject, the method includes administering to a subject in need thereof a therapeutically effective amount of a compound represented by general Formula I, or a pharmaceutical composition comprising the same.

According to some embodiments of the present invention, intraocular pressure (IOP) is associated with glaucoma, low-tension glaucoma and normal-tension glaucoma. Hence, the compounds, compositions and medicaments presented herein are useful in treating, without limitation, glaucoma, low-tension glaucoma and normal-tension glaucoma.

As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

As used herein, the phrase “therapeutically effective amount” describes an amount of the polymer being administered which will relieve to some extent one or more of the symptoms of the condition being treated.

In some embodiments, the concentration of the compounds presented herein in the pharmaceutical compositions or medicaments presented herein, is in the range of about 1 to about 5,000 μg/ml of composition, preferably from about 80 to about 800 ag/ml and the formulation is preferably applied in one to four doses per day wherein each dose contains about 1 to 125 μg of the compound, or from about 2 to about 20 μg of the compound.

Co-Administration with Other IOP Reducing Agents:

In the experimental section presented below, it has been demonstrated that a combination of a compound according to some embodiments of the present invention and Latanoprost resulted in a notable increase in the duration of the effects for the combination compared to the individually administered agents.

In the context of some embodiments of the present invention, an active agent which is not a compound represented by general Formula I or general Formula III, is referred to herein as “another agent” or “other agent”.

According to an aspect of some embodiments of the present invention, there is provided a pharmaceutical composition which includes as active ingredients at least one other agent (active ingredient) which is in use in reducing IOP and is not a compound represented by general Formula I or general Formula III, a compound represented by general Formula I or general Formula III, and a pharmaceutically acceptable carrier.

Non-limiting examples of active ingredients which are in use in reducing IOP and which are referred to herein other agents, include prostaglandin analogs, β-blockers, adrenergic agents, α2-adrenergic receptor agonists, miotic agents, carbonic anhydrase inhibitors and cholinergic agonists.

Non-limiting examples of prostaglandin analogs include Latanoprost (Xalatan), Bimatoprost (Lumigan) and Travoprost (Travatan).

Non-limiting examples of β-blockers include Timolol (Timoptic) and Betaxolol (Betoptic).

A non-limiting example of adrenergic agents is Brimonidine (Alphagan).

A non-limiting example of miotic agents is Pilocarpine (Isoptocarpine, Pilocar).

Non-limiting examples of carbonic anhydrase inhibitors include Dorzolamide (Trusopt), Brinzolamide (Azopt) and Acetazolamide (Diamox).

Non-limiting examples of cholinergic agonists include carbachol (Miostat), echothiophate (Phospholine) and pilocarpine (Isopto Carpine, Pilopine).

Administration of more than one active agent to a subject is generally known in the art as co-administration. The term “co-administration” as used herein, refers to a concomitant administration of more than one active agent (active ingredient) to a subject, whereas in the context of embodiments presented herein, the term “concomitant” means that the co-administered active agents are present in the subject (PK), or otherwise exert an effect (PD), at similar, identical, consecutive or partially overlapping periods of time.

In the context of co-administration of more than one active agent, the terms “substantially simultaneous” and “rapid succession” correspond to the term “concomitant” as used herein, namely meaning that the period of time between a first administration and a second administration of more than one active agent is sufficiently short to be regarded as a single administration event, and/or a number of administrations of different active agents takes place within 5-60 minutes or less. Optionally, each administration in such “rapid succession” delivers to the user a different amount or composition of one or more pharmaceutically active agents. Alternatively, two or more of the administrations provide the same composition and amount of the one or more pharmaceutically active agents. In some embodiments, the later administration of the second active agent is performed at such timing that the first active agent of a previous administration within the same rapid succession does not yet have a significant pharmacodynamic effect or before it can be measured (pharmacokynetically) by, e.g., blood concentration thereof the first active agent.

According to some embodiments of the present invention, the co-administration of at least one other agent which is in use in reducing IOP and is not a compound represented by general Formula I or general Formula III, and a compound represented by general Formula I or general Formula III, exhibits a potentiating effect, namely that the effect of the co-administered active ingredients is greater in at least one parameter, such as magnitude or duration, compared to the effects exhibited by each of the active ingredients when administered alone (separately).

Accordingly, there is provided a use of at least one other agent which is in use in reducing IOP, and a compound represented by general Formula I or general Formula III, for the manufacture of a medicament for reducing IOP.

According to an aspect of some embodiments of the present invention, there is provided a method of reducing IOP in a subject in need thereof, the method includes co-administering to the subject a therapeutically effective amount of at least one other agent which is in use in reducing IOP, and a therapeutically effective amount of a compound represented by general Formula I or general Formula III.

According to an aspect of some embodiments of the present invention, there is provided a method of reducing IOP in a subject in need thereof, the method includes co-administering to the subject a synergistically effective amount of at least one other agent which is in use in reducing IOP, and a synergistically effective amount of a compound represented by general Formula I or general Formula III. It is noted herein that a synergistically effective amount is also a therapeutically effective amount in the sense of providing the desired therapeutic effect, and is smaller than the therapeutically effective amount of a singly-administered active ingredient.

In the context of the herein provided uses and method of co-administration of other agents and the compounds presented herein, the other agent is a prostaglandin analog.

In the context of the herein provided uses and method of co-administration of other agents and the compounds presented herein, the other agent is a β-blocker.

In the context of the herein provided uses and method of co-administration of other agents and the compounds presented herein, the other agent is an adrenergic agent.

In the context of the herein provided uses and method of co-administration of other agents and the compounds presented herein, the other agent is an α2-adrenergic receptor agonist.

In the context of the herein provided uses and method of co-administration of other agents and the compounds presented herein, the other agent is a miotic agent.

In the context of the herein provided uses and method of co-administration of other agents and the compounds presented herein, the other agent is a carbonic anhydrase inhibitor.

In the context of the herein provided uses and method of co-administration of other agents and the compounds presented herein, the other agent is a cholinergic agonist.

Cardiotonic Agent:

According to some embodiments, the selectivity of the compounds presented herein can be utilized in the treatment of other medical conditions. For example, an α2-selective inhibitor, such as the compounds presented herein and represented by general Formula I, can be used as an effective cardiotonic agent, with reduced cardiotoxicity, compared to known agents such as digoxin.

According to an aspect of embodiments of the present invention, there is provided a method of treating a heart condition which is carried out by administering to a subject in need thereof a therapeutically effective amount of a compound represented by general Formula I.

According to an aspect of embodiments of the present invention, there is provided a pharmaceutical composition which includes as an active ingredient a compound represented by general Formula I and a pharmaceutically acceptable carrier, identified in print, or on a packaging material, for use in a treatment of a heart condition.

According to an aspect of embodiments of the present invention, there is provided a uses of a compound represented by general Formula I or a pharmaceutical composition comprising the same, for the manufacture of a medicament for treating a heart condition.

Examples of heart conditions which are relevant in the context of embodiments of the resent invention, include, without limitation, atrial fibrillation, atrial flutter, mitral stenosis, chronic heart failure and congestive heart failure.

In some embodiments, the present invention provides cardiotonic compositions comprising a therapeutically effective amount of the compounds represented by general Formula I, or a pharmaceutical composition comprising the same. In accordance with such embodiments, the compounds may be formulated for oral, buccal, topical, intravenous, parenteral or rectal administration.

A compound represented by general Formula I or a compound represented by general Formula III can be used according to embodiments the present invention to treat a heart condition in combination with one or more other drugs for treating a heart a condition, such as, but not limited to, selective and nonselective β-blocker agents, anticoagulation agents, angiotensin-converting-enzyme inhibitors (ACE inhibitors) and angiotensin II receptor antagonists.

According to an aspect of embodiments of the present invention, there is provided a method of treating a heart condition in a subject in need thereof, which includes co-administering to the subject a therapeutically effective amount of an agent selected from the group consisting of a β-blocker, an anticoagulation agent, an angiotensin-converting-enzyme inhibitor and an angiotensin II receptor antagonist; and

a compound represented by Formula III, as these are described herein, and a pharmaceutically acceptable carrier.

According to an aspect of embodiments of the present invention, there is provided a pharmaceutical composition that includes, as active ingredients, at least one ingredient selected from the group consisting of a β-blocker, an anticoagulation agent, an angiotensin-converting-enzyme inhibitor and an angiotensin II receptor antagonist; and a compound represented by Formula III, as these are described herein.

According to an aspect of embodiments of the present invention, there is provided a use of an agent selected from the group consisting of a β-blocker, an anticoagulation agent, an angiotensin-converting-enzyme inhibitor and an angiotensin II receptor antagonist, and a compound represented by Formula III, as these are described herein, for the manufacture of a medicament for treating a heart condition.

Nonselective β-blocker agents include propranolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutolol, pindolol, sotalol, timolol, eucommia bark (herb); β1-selective agents include acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, esmolol, metoprolol, nebivolol; β2-selective agents include butaxamine and ICI-118,551; and β3-selective agents include SR 59230A.

Anticoagulation agents include heparin, dicumarol, coumadin (warfarin) and aspirin.

ACE inhibitors include captopril, zofenopril, enalapril (vasotec, renitec), ramipril (altace, prilace, ramace, ramiwin, triatec, tritace), quinapril (accupril), perindopril (coversyl, aceon, perindo), lisinopril (listril, lopril, novatec, prinivil, zestril), benazepril (lotensin), imidapril (tanatril), trandolapril (mavik, odrik, gopten), cilazapril (inhibace) and fosinopril (fositen, monopril).

Angiotensin II receptor antagonists include losartan, EXP 3174, candesartan, valsartan, irbesartan, telmisartan, eprosartan, olmesartan, azilsartan and fimasartan.

According to some embodiments, the compound represented by general Formula I or the compound represented by general Formula III, according to embodiments of the present invention, and any one of the other drugs for treating a heart a condition can be co-formulated in a single composition, or be formulated into individual compositions.

High-Risk Pharmacokinetics:

When plasma concentrations of active drug depend exclusively on a single metabolic pathway, any condition that inhibits that pathway (be it disease-related, genetic, or due to a drug interaction) can lead to dramatic changes in drug concentrations and marked variability in drug action. This problem of high-risk pharmacokinetics is especially pronounced in drug elimination that relies on a single pathway. In this case, inhibition of the elimination pathway leads to striking elevation of drug concentration. For drugs with a narrow therapeutic window, this leads to an increased likelihood of dose-related toxicity. An example is digoxin, whose elimination is dependent on P-glycoprotein; many drugs inhibit P-glycoprotein activity (amiodarone, quinidine, erythromycin, cyclosporine, itraconazole) and coadministration of these with digoxin reduces digoxin clearance, and increases toxicity unless maintenance doses are lowered. Drugs with a high risk of generating pharmacokinetic interactions with digoxin include antacids and bile acid sequestrants, which can cause reduced absorption; inhibitors of CYPs and of P-glycoprotein such as amiodarone, quinidine, amiodarone, verapamil, cyclosporine, itraconazole and erythromycin which can cause decreased clearance.

Since compounds encompassed under Formula III are in essence digoxin derivatives that exhibit lower digoxin toxicity, these compounds can be used with any of the drugs described above which exhibit adverse drug-drug interaction with digoxin.

According to an aspect of embodiments of the present invention, there is provided use of the compounds represented by general Formula III for the treatment of a medical condition or a combination of medical conditions, with is treatable by digoxin and a drug having an adverse interaction with digoxin.

Prodrugs:

Prodrugs corresponding to the compounds presented herein are contemplated in the context of the resent invention in order to provide active agents that exhibit improved pharmacokinetic and/or pharmacodynamics profile, and/or a reduction in adverse effects. As known in the art, a prodrug is typically a chemical derivative of the corresponding drug, being chemically modified such that a naturally occurring metabolic pathway in the subject's system can convert it to the parent drug molecule during the time in which the prodrug/drug is still present in the system. For instance, drugs that exhibit poor absorption due to high hydrophobicity, can be modified to exhibit improved bioavailability by introducing chemical functionalities that increase the solubility of the prodrug comparted to the parent active compound. In other cases where the active compound exhibits adverse effects in the GI-tract, the prodrug is a modified parent active compound that is metabolized by enzymes back to the parent compound substantially at the target tissues, organs or cells.

In the context of some embodiments of the present invention, the prodrugs are metabolized to afford the corresponding (parent) compound by naturally occurring metabolic agents, such as enzymes. In some embodiments, the chemical group of the prodrug is bioliable or biodegradable, such as in the case of an ester, which can be hydrolyzed by esterases to afford a hydroxyl of the parent compound.

According to some embodiments, the compounds presented herein exhibit several structural positions that can offer potential locations for chemical modifications on route to becoming suitable prodrugs. For example, a digoxin skeleton exhibits four hydroxyl groups which can potentially be converted into, e.g., esters, under various conditions. Similarly, the digitoxin skeleton exhibits three hydroxyl moieties. Since each hydroxyl is located at a different chemical environment, is it chemically converted under varying conditions, thereby allowing the production of a single or a multiple hydroxyl-to-ester conversion. Thus, in the case of digoxin, a mono-, a bis-, a tris- or a tetra-modified parent compound can be afforded, offering a variety of prodrugs of the same patent compound, each exhibiting a different pharmacokinetic/pharmacodynamics profile.

In addition, R and R′ substituents (see, Formula I and Formula III respectively) may exhibit a chemical position which is readily converted into another, bioliable (biodegradable) moiety, and thus can be used to afford prodrugs from the corresponding parent compound.

According to some embodiments, prodrugs of the compounds having the general Formula I or general Formula III, are represented by general Formula V:

wherein X is H or PD₃, and each of PD₁-PD₄ is H or independently represents a modified hydroxyl group turned into a bioliable functionality, provided that at least one of PD₁-PD₄ is not H.

In some embodiments, the compounds presented herein are converted into a prodrug by modifying any one of the hydroxyl groups on the digoxin or digitoxin moiety of the compounds into any one of a methoxymethyl ether, a tetrahydropyranyl ether, a t-butyl ether, an allyl ether, a benzyl ether, a t-butyldimethylsilyl ether, a t-butyldiphenylsilyl ether, an acetic acid ester (Ac), ethyl, propyl, butyl, t-butyl or pivalic acid ester and a benzoic acid ester, in any combination. Each of the converted hydroxyl functionalities may be metabolized (biodegraded) back to the parent hydroxyl by one or more metabolic and/or enzymatic systems.

According to some embodiments, any one of PD₁-PD₄ in general Formula V is selected from the group consisting of a methoxymethyl ether, a tetrahydropyranyl ether, a t-butyl ether, an allyl ether, a benzyl ether, a t-butyldimethylsilyl ether, a t-butyldiphenylsilyl ether, an acetic acid ester (Ac), ethyl, propyl, butyl, t-butyl or pivalic acid ester and a benzoic acid ester.

As can be seen in the Examples section that follows below, the exemplary digoxin-derived compound DcB, according to some embodiments of the present invention, has been converted successfully into the corresponding bit-acetyl (BisAcDcB) prodrug and the tris-acetyl (TrisAcDcB) prodrug (see, Example 6).

Isolation and Use of Na,K-ATPase Isoforms:

According to an aspect of some embodiments of the present invention, there is provided a method of determining an apparent affinity of the compound represented by general Formula I to at least one isoform of Na,K-ATPase, the method includes contacting an isoform of Na,K-ATPase with the compound in an activity measurement setup and determining the apparent affinity of the compound to the isoform.

According to an aspect of some embodiments of the present invention, there is provided a method of isolating an isoform of Na,K-ATPase of a mammal, the method includes:

transforming yeast cells with a clone that comprises an α chain sequence and a β chain sequence of the Na,K-ATPase;

expressing the clone in the yeast cells; and

isolating the isoform,

wherein:

the α chain sequence is selected from the group consisting of α1, α2, α3 and α4; and

the β chain sequence is selected from the group consisting of β1, β2 and β3.

In some embodiments, the isolated isoform is α2β2.

In some embodiments, the isolated isoform is α2β3.

According to an aspect of some embodiments of the present invention, there is provided an isolated α2β2 isoform of Na,K-ATPase of a mammal having at least 70% purity.

According to an aspect of some embodiments of the present invention, there is provided an isolated α2β3 isoform of Na,K-ATPase of a mammal having at least 70% purity.

The yeast-expressed α/β subunits have distinct levels of glycosylation compared to those in the human-expressed subunits. The distinct yeast-expressed glycosylation pattern does not substantially affect the activity of the enzyme but may increase its stability.

According to an aspect of some embodiments of the present invention, there is provided an isolated α2β2 isoform of Na,K-ATPase of a mammal having a yeast-characterizing glycosylation pattern.

According to an aspect of some embodiments of the present invention, there is provided an isolated α2β3 isoform of Na,K-ATPase of a mammal having a yeast-characterizing glycosylation pattern.

In some embodiments, the isolated isoforms presented herein have an amino acid sequence which identical, substantially similar or derived from any one of the isoform of human of Na,K-ATPase.

SEQ ID Nos. Description SEQ ID No. Amino acid sequence of α1 subunit of human Na,K-ATPase (P05023) 1 Amino acid sequence of α2 subunit of human Na,K-ATPase (P50993) 2 Amino acid sequence of α3 subunit of human Na,K-ATPase (P13637) 3 Amino acid sequence of α4 subunit of human Na,K-ATPase (Q13733) 4 Amino acid sequence of β1 subunit of human Na,K-ATPase (P05026) 5 Amino acid sequence of HIS tagged β1 subunit of human Na,K-ATPase (P05026) 6 Amino acid sequence of β2 subunit of human Na,K-ATPase (P14415) 7 Amino acid sequence of HIS tagged β1 subunit of human Na,K-ATPase (P14415) 8 Amino acid sequence of β3 subunit of human Na,K-ATPase (P54709) 9 Amino acid sequence of HIS tagged β3 subunit of human Na,K-ATPase (P54709) 10 Nucleotide sequence of α1 subunit of human Na,K-ATPase (ATP1A1) 11 Nucleotide sequence of α2 subunit of human Na,K-ATPase (ATP1A2) 12 Nucleotide sequence of α3 subunit of human Na,K-ATPase (ATP1A3) 13 Nucleotide sequence of α4 subunit of human Na,K-ATPase (ATP1A4) 14 Nucleotide sequence of HIS tagged β1 subunit of human Na,K-ATPase (ATP1B1) 15 Nucleotide sequence of HIS tagged β2 subunit of human Na,K-ATPase (ATP1B2) 16 Nucleotide sequence of HIS tagged β3 subunit of human Na,K-ATPase (ATP1B3) 17 Nucleotide sequence of HIS tagged human FXYD1 18 Amino acid sequence of HIS tagged human FXYD1 19

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof. Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is expected that during the life of a patent maturing from this application many relevant inhibitors exhibiting selectivity towards α2-containing isoforms of Na,K-ATPase, as defined herein, will be uncovered and the scope of this term is intended to include all such new selective inhibitors a priori.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

Example 1 Compound Synthesis

Compounds represented by general Formula I, according to embodiments of the present invention, were synthesized as a series of perhydro-1-4-oxazepine derivatives of the third digitoxose of digoxin or digitoxin following a general procedure described elsewhere [Adamczyk, M. et al., Steroids, 1995, 60(11), pp. 753-758] by selective oxidation with NaIO₄ and reductive amination with NaCNBH₃. The compounds were purified by HPLC and masses, ¹H-NMR and ¹³C-NMR spectra were determined to verify both correctness of the structure and purity.

The structure and names based on of the amine substituents (R—NH₂), as well as their theoretical and measured masses, are presented in Table 1 below, which includes other digoxin derivatives with non-cyclic moieties (last eight compounds in Table 1).

TABLE 1 Theoretical Measured mass R Name mass (with Na+ ion) cyclopropyl DcP 803.48 826.45 methylcyclopropane DMcP 817.50 840.44 ethylcyclopropane DEcP 831.51 854.58 cyclobutyl DcB 817.50 840.43 cyclopentyl DcPe 831.51 854.51 cyclohexyl DcHe 845.53 868.65 benzyl DBz 853.50 876.44 methyl(3,3- DMDMcB 859.54 882.59 dimethylcyclobutane) methylcyclopropane DtxMcP 801.50 824.48 cyclobutyl DtxcB 801.50 824.44 methyl DMe 777.47 800.57 ethyl DEt 791.48 814.52 2,2,2-trifluoroethyl DCF₃ 845.45 868.14 propyl DP 805.50 828.27 iso-propyl DiP 805.50 828.41 iso-butyl DiB 819.51 842.66 tert-butyl DtB 819.51 842.41 methyl(trimethylsilyl) DTMS 849.51 872.77 (methylsulfonyl)methyl DMSM 855.44 878.49 (methylsulfonyl)ethyl DESM 869.46 892.29 (sulfonamide)ethyl DESA 870.45 893.47 azetidinyl DAz 818.49 841.55 oxetanyl DOx 819.48 842.41 thietanyl DTh 835.45 858.37 histaminyl DHis 857.50 880.45 (N,N-dimethylamine)ethyl DEDA 834.52 857.62 aminoethyl DED 806.49 829.55 (methylsulfonyl)methyl DMSM 855.44 878.49

Names of compounds encompassed under Formula I wherein X=OH (digoxin derivatives) include Dcp, DMcP, DEcP, DcB, DcB, DcB, DcB and DMDMcB; names of compounds encompassed under Formula I wherein X=H (digitoxin derivatives) include, DtxMcP and DtxcB.

Example 2 Expression, Purification and Characterization of Recombinant Human Na,K-ATPase Isolated Isoforms

Plasmid construction for the expression of α1β1, α2β1, α2β2 and α2β3 Na,K-ATPase was conducted by generation of pHil-D2 expression vector containing cDNA of human α₁ and His10 tagged human β₁ was described previously [Lifshitz Y, et al., 2007, Biochemistry, 46(51), pp. 14937-14950]. cDNA of human β₂ and β₃ in pSD5 were a gift from K. Geering, University Lausanne, Switzerland. Open reading frames and flanking regions of human β₂ and human β₃ were amplified by PCR using primers containing BglII and SalI cleavage sites. The resulting fragments were subcloned into pHil-D2-hα₂/His10-pβ₁ to create pHil-D2-hα₂/His10-hβ₂ and pHil-D2-hα₂/His10-hβ₃, respectively. Correct integration and sequence was confirmed by sequencing.

Yeasts were grown in BMG (100 mM potassium phosphate pH 6, 1.34% yeast nitrogen base, 4×10⁻⁵% biotin, 0.3% glycerol) to OD 6-8 and expression was induced in BMM (100 mM potassium phosphate pH 6, 1.34% yeast nitrogen base, 4×10⁻⁵% biotin, 0.5% methanol added daily).

Pichia pastoris transformation, yeast growth, membrane preparation and His-tag purification of recombinant human α1β1, α2β1, α2β2 and α2β3 Na,K-ATPase were carried out essentially as described previously [Katz A, et al., 2010, J Biol Chem, 285(25), pp. 19582-19592; and Katz A, et al., 2014, J Biol Chem, 289(30), pp. 21153-21162].

Expression and purification of α1β1, α2β1, α2β2 and α2β3 complexes was conducted in small scale whole cell lysates that were prepared as describe in Loayza, D. et al., Mol. Cell. Biol., 1998, 18, p. 779-789. Yeast membrane production and expression of recombinant Na,K-ATPase as detergent soluble complexes was performed as described in Habeck, M. et al., J. Biol. Chem., 2015, 290, pp. 4829-4842, using modified lipid content (C12E8, 0.1 mg/ml; SOPS, 0.07 mg/ml; cholesterol, 0.01 mg/ml).

Na,K-ATPase α₁β₁, α₂β₁, α₂β₂ and α₂β₃ complexes were reconstituted with FXYD1 and purified in a mixture of 0.1 mg/ml C12E8, 0.07 mg/ml SOPS and 0.01 mg/ml cholesterol.

The purified isoform complexes (0.3-0.5 mg/ml) were eluted from the BD-Talon beads in a solution containing Imidazole 170 mM, NaCl 100 mM; Tricine.HCl 20 mM pH 7.4; C12E8, 0.1 mg/ml; SOPS 0.07 mg/ml, cholesterol 0.01 mg/ml, glycerol 25%, by gravity-column. The proteins were stored at −80° C. Protein purity was determined by gel electrophoresis and protein concentration was determined with BCA (B9643 Sigma).

The specific Na,K-ATPase activity was highest for α₁β₁ (16.4±0.7 μmol/mg/min) followed by α₂β₁ (10.9±0.6) and α₂β₃ (10.7±1.9). The α₂β₂ isoform had the lowest activity (8.4±1.4). The second significantly different parameter is the apparent K⁺-affinity. K_(0.5) K⁺ for α₂β₁ was 2.7±0.14 mM compared to 1.47±0.06 mM for α₁β₁. α₂β₂ and α₂β₃ had an even lower affinity than α₂β₁ with apparent K_(0.5) values of 7.4±0.19 mM and 6.4±0.50 mM, respectively. Na-titrations revealed that the affinity for Na⁺-ions was not different between α₁β₁ and α₂β₁ whereas α₂β₂ and α₂β₃ had a somewhat higher Na-affinity.

The reduced apparent affinity for K⁺ together with an increased affinity for Na⁺ indicates that the conformational equilibrium of α₂β₂ and α₂β₃ might be shifted towards E1. In order to test this hypothesis, the apparent affinity of vanadate was determined for all four isoform complexes. Vanadate is a phosphate analogue that binds to the E2 conformation, mimicking the transition state E2PK₂ during dephosphorylation, thus inhibiting the enzyme. All three α2 isoforms had a lower vanadate affinity compared to α₁β₁ (0.48 μM). α₂β₂ had the lowest vanadate affinity (34 μM) followed by α₂β₃ (19 μM) and α₂β₁ (3.5 μM). Thus, the order of inhibition by vanadate equals the order of potassium activation (K_(0.5)K⁺ and K_(i) vanadate α₁β₁<α₂β₁<α₂β₃<α₂β₃) supporting the hypothesis proposed above.

Example 3 Selective Inhibition Assays of Isolated Na,K-ATPase

To screen for isoform selectivity of the digoxin derivatives we compared inhibition of Na,K-ATPase activity of purified detergent-soluble human isoform complexes α1β1FXYD1, α2β1FXYD1, α2β2FXYD1 and α2β3FXYD1. Although all the preparations and assays were conducted with FXYD1 in order to stabilize the complexes, the FXYD1 suffix is omitted in naming of isoform complexes for simplicity.

Na,K-ATPase activity of α/βFXYD1 complexes was measured over one hour at 37° C. in a medium containing 130 mM NaCl, 5 mM KCl, 3 mM MgCl₂, 1 mM EGTA, 25 mM Histidine, pH 7.4 and 1 mM ATP using the PiColor Lock gold malachite green assay (Inova Biosciences).

The Na,K-ATPase activities were α1β1, 21.5±5.3 moles/min/mg; α2β1, 18.7±1.8 moles/min/mg, and α2β3, 10.7±1.9 moles/min/mg protein. As discussed below, an important kinetic property in relation to inhibition by cardiac glycosides is K_(0.5) for activation by K: α1β1—1.25±0.05 mM, α2β1—2.7±0.14 mM and α2β3 6.4±0.50 mM, respectively.

Selectivity of the compounds for various isolated isoforms of human Na,K-ATPase was determined essentially as described before [Katz, A. et al., J Biol Chem., 2010, 285(25), pp. 19582-19592].

ATPase activity assays as well as titrations with NaCl, KCl and vanadate were performed as described in Lifshitz-2007 and Loayza-1998 using PiColorLock™ malachite green assay (Inova Bioscience). Inhibitor assays were performed as described in Katz-2010. [³H]ouabain binding and K⁺-[³H]digoxin displacement assays were performed as described in Katz-2010.

The percent inhibition VCG/V0 was calculated and Ki values were obtained by fitting the data to the function VCG/V0=Ki/([CG]+Ki)+c (CG stands for cardiac glycoside). Inhibition was estimated in 3-5 separate experiments and average Ki values±standard error of the mean (SEM) were calculated. The ratios Ki α1β1/α2β1, α1β1/α2β2 and α1β1/α2β3 was calculated for each compound.

Table 2 shows the Ki values and selectivity ratios (Ki α1β1:α2β1, Ki α1β1:α2β2 and α1β1:α2β3) for inhibition of Na,K-ATPase activity of compounds according to embodiments of the present invention, as well as some digoxin derivatives having a non-cyclic moiety, compared to digoxin and digitoxin. Table 2 is arbitrarily sorted according to column “Ki ratio α1β1/α2β3” marked by “*”

TABLE 2 No. of C Selectivity atoms in Ki in nM ± SEM Ki ratio Ki ratio *Ki ratio Compound Name R α1β1 α2β1 α2β2 α2β3 α1β1/α2β1 α1β1/α2β2 α1β1/α2β3 DcB (cyclic) 4 135 ± 11    8 ± 1.25  6 ± 1    4 ± 0.15 16.9 22.2 33.6 DMcP (cyclic) 4  95.8 ± 13.7 18.3 ± 1.6  8.0 ± 0.8  4.3 ± 0.6 5.2 12 22.2 DESM (sulfonyl) 2 464 ± 14 49.2 ± 1.9 31.7 ± 3.2 24.7 ± 2.1 9.4 14.6 18.8 DiB 4   92 ± 8.9 20.6 ± 1.4   10 ± 0.8  5.8 ± 0.6 4.4 9.0 16 DESA (sulfonamide) 2 301 ± 23 38.9 ± 2.2 31.5 ± 4.4 20.1 ± 0.9 7.7 9.5 15 DiP 3   149 ± 20.7 28.9 ± 1.7 16.7 ± 1.9 10.3 ± 1.8 5.1 8.9 14.4 DcP (cyclic) 3  109 ± 6.2  14.6 ± 11.6 13.0 ± 1.3  8.1 ± 1.36 7.5 8.5 13.4 DEcP (cyclic) 5 86.1 ± 7   14.3 ± 2   12.1 ± 1.9 7.2 ± 1  6.02 7.1 12.02 DMSM (sulfonyl) 3  944 ± 123  137 ± 9.8  123 ± 7.3   89 ± 8.7 6.9 7.7 10.6 DBz (cyclic) 6  57.9 ± 15.5 10.1 ± 2.2  6.8 ± 1.2  5.6 ± 1.6 5.7 8.5 10.3 DcH (cyclic) 6 70.4 ± 4.1 15.2 ± 3.7 15.3 ± 2.9 11.7 ± 4.5 4.6 4.6 10.1 DCF₃ 2   119 ± 15.0 28.6 ± 0.9 18.1 ± 1.9 12.4 ± 1.5 4.1 6.5 9.6 DEt 2 137.9 ± 12.6 23.2 ± 0.9 16.4 ± 1.6  14.4 ± 1.27 5.9 8.3 9.5 DMe 1  103 ± 5.6 15.3 ± 1.2 20.36 ± 1.8  10.8 ± 0.6 6.7 5.1 9.5 DP 3 87.7 ± 7.9  18.3 ± 1.68 10.5 ± 1.8  9.8 ± 1.1 4.8 8.3 8.8 DtB 4   135 ± 12.1 21.6 ± 5.6 18.4 ± 1.1  16.3 ± 0.28 6.2 7.3 8.2 DMDMcB (cyclic) 7 31.6 ± 0.5  8.6 ± 1.4  5.1 ± 0.5  3.9 ± 0.7 3.69 6.16 8.19 DtxcB (cyclic) 4 30.7 ± 7.2  5.4 ± 0.5  5.3 ± 0.8  4.3 ± 0.6 5.6 5.8 7.1 DtxMcP (cyclic) 4 25 ± 4  4.2 ± 0.4  5.4 ± 1.2  3.7 ± 0.5 5.9 4.6 6.7 DTMS (sily1) 4  72.6 ± 17.6 24.3 ± 4.0 14.3 ± 1.3 11.1 ± 1.6 3 5.1 6.5 Digoxin 0   268 ± 13.8 58.7 ± 5.4   58 ± 1.9 42.8 ± 3.0 4.5 4.6 6.2 DcPe (cyclic) 5 138 ± 21 33.4 ± 7.5  33.5 ± 11.9 27.6 ± 9.5 4.1 4.1 5 Digitoxin 0   89 ± 15.8 29.5 ± 2.7 40.7 ± 6.7 28.8 ± 5.9 3 2.1 3.1

As can be seen in Table 2, some compounds show an even greater selectivity ratio towards α2β2 and α2β3, particularly α2β3. Based on the ratios of Ki values, several derivatives show significantly improved selectivity for α2β3 compared to α2β1 over α1β1, in particular DMcP and DcB. DMcP and DcB show exceptional selectivity for α2β2 and α2β3 over α1β3 of 22-fold and 33-fold, respectively, and very low Ki values for inhibition of α2β3 (Ki about 4 nM). The full inhibition curves of DMcP and DcB emphasize the extent of the difference between α1β1 and α2β3.

Bearing in mind the large differences in K_(0.5) potassium ions, a point to be aware of in analyzing the results presented in Table 2 is the well-known K-cardiac glycoside antagonism. Digoxin itself has moderate selectivity for α2β1 over α1β1 (about 4-fold) and the data in Table 2 shows increased selectivity for α2β3 over α1β1 (about 6-fold). The α2β1-selectivity of digoxin is attributed to a combination of increased binding affinity for α2 over α1 and also reduced K-digoxin antagonism in the Na,K-ATPase reaction conditions (with K, 5 mM). Similarly, the increased selectivity of digoxin for α2β3 compared to α2β1 is attributable to reduced K-cardiac glycoside antagonism due to the higher K_(0.5) K of α2β3 compared to α2β1.

The compounds presented herein show a notable increase of the ratio of Ki's α1β1:α2β3 compared to α1β1:α2β1, which must be partly due to the reduced K-cardiac glycoside antagonism. However, the difference for the most α2β3-selective compounds is significantly greater than that for digoxin and cannot be explained only by this factor. In particular, there is a distinct structural effect in that the maximal α2β3-selectivity is seen for R-substituents with cyclic moiety, as well as those with four carbon atoms. In the case of DMcP and DcB, the Ki for α1β1 is about 2-3-fold lower than for digoxin itself but the Ki for α2β3 is about 10-fold lower than for digoxin, thus raising selectivity for α2β3 over α1β1 to more than 22-fold and more than 33-fold, respectively. It is also noticeable that for derivatives with five or a higher number of carbon atoms in the R-substituents, such as DEcP, DcPe, DcH, DBz and DDMcB, although the Ki values for all isoforms are all lower than for digoxin itself, the selectivity for α2β3 over α1β1, 10-12-fold, is also lower than the selectivity observed in four carbon R-substituents, DMcP and DcB. The same is true for compounds with R-substituent three carbons or shorter.

Another structural insight comes from the results obtained for two digitoxin derivatives (DtxMcP and DtxcB) compared with the digoxin derivatives DMcP and DcB. As seen in Table 2, the Ki values are relatively low for all three isoform complexes α1β1, α2β1 and α2β3, being reduced about 10-fold compared to digoxin. Consequently, these digitoxin derivatives showed little increase in selectivity for α2β3 compared to digoxin itself. Thus, the absence of a single OH group in position 12 of the steroid moiety of digitoxin reduced the effect of the sugar modification on α2β3-selectivity.

Thus, modification of the third digitoxose moiety of digoxin, but not of digitoxin, with cyclic substituents such as McP and cB confers notable α2β3-selectivity, presumably due to selective interaction with β3.

Example 4 Inhibition of Na,K-ATPase Activity in Permeabilized Bovine NPE Cells

In order to prove the concept of selective inhibition of the compounds presented herein in Na,K-pumps of native ciliary epithelium, inhibition assays using Na,K-ATPase in NPE cells isolated from the ciliary body dissected out of bovine eyes were conducted.

Isolation of ciliary epithelium PE and NPE cells was carried out as previously described [Edelman, J. L. et al., 1994, Am J Physiol, 266(5 Pt 1), pp. C₁₂₁₀-1221], with some modifications, using 15-20 fresh bovine eyes. Ciliary bodies were isolated from bovine eyes and washed in ringer solution. The tissue was treated with trypsin and homogenized followed by separation on a density gradient of Metrizamide, which separates between the NPE and PE cells.

Using isolated human isoforms to calibrate the response of the antibodies, NPE cell lysates were shown to contain about 70% α2 and 30% α1, while PE were shown to contain about 90% α1 and 5-10% α2. After unmasking the Na,K-ATPase by treating the cells with alamethicin, Na,K-ATPase activity was measured and was found to be 0.195±0.027 and 0.035±0.008 nmoles/mg protein/min in NPE and PE cells, respectively. Ouabain-sensitive fractions of total ATPase activity were about 65% and 35% for NPE and PE cells, respectively.

To determine Na,K-ATPase activity in the cells, the cells were incubated with 0.8 mg/ml alamecithin for 30 minutes at room temperature prior to transfer to the reaction medium containing 130 mM NaCl, 5 mM KCl, 3 mM MgCl₂, 25 mM histidine, pH 7.4, 1 mM EGTA, 1 mM sodium azide, 0.5 mM ATP, and were then incubated for 45 minutes at 37° C., with or without the tested inhibitors as indicated, or 0.5 mM ouabain to determine the ouabain insensitive ATPase activity. The data was fitted to one or two sites inhibition model.

Table 3 presents Ki values for inhibition of NPE Na,K-ATPase activity by digoxin, DMe, DMcP and DcB, fitted to a single site inhibition model.

TABLE 3 Compound Ki, nM ± SEM n 1 site model Digoxin 91.7 ± 10.2 4 DMe 15.6 ± 1.3  4 DMcP 7.9 ± 2.2 5 DcB 17.3 ± 2.5  4 2 sites model DcB Ki α2 6.9 ± 2 Ki α1 151 ± 7.6 4 (A α2 0.66 ± 0.090 A α1 0.34 ± 0.097)

As can be seen in Table 3, the Ki values of the derivatives are all lower than that of digoxin. Since NPE cells contain about 70% α2 and 30% α1, Na,K-ATPase activity and inhibition should reflect the properties of the isoform mixture. Indeed, the detailed inhibition curve for the most α2β3-selective compound, DcB, was fitted better by a two site model, compared to fitting according to a one site model.

As can be seen in Table 3, the two site model provides the best fit parameters of 66% α2, Ki 6.9±2 nM; 34% α1, Ki 151±7.6 nM (Ki α1/α2=22), which are quite close to the proportions of α2:α1 estimated in the immunoassays, and the selectivity ratio Ki α1β1/α2β3 is about 33.

Thus, it can be concluded that the selectivity properties of the digoxin derivative DcB observed with purified human isoforms is corroborated by the results obtained using intact NPE cells.

Example 5 Reduction of Intraocular Pressure

These experiments examined the effects of topically administered α2β3-selective digoxin derivatives, according to some embodiments of the present invention, DiB, DMcP and DcB, on IOP in rabbits. Due to the lower Ki for inhibition of α2β3, compared to digoxin, and the high hydrophobicity, these compounds were predicted to both permeate the cornea well and efficiently inhibit the α2β3 in the NPE ciliary epithelium, thus reducing inflow of aqueous humour and IOP.

New Zealand white rabbits (3-3.5 kg) about 1 year old, of either sex, were housed in pairs in cage in animal room conditions on a reversed, 12-hour dark/light cycle. For the experiments the animals were transferred to rabbit restrainers in a quiet and calm atmosphere. No ocular abnormalities were detected prior or during the experiments.

IOP measurements were made with a pneumatonometer (Model 30, Reichert technologies) either after raising IOP with 4-aminopyridine (4AP; 1 drop 40 mg/ml), or on basal IOP after addition of one drop of 1 mM solution of digoxin derivatives to the right eye (RE) and one drop of PBS to the left eye (LE) that served as control.

For comparison of effects of digoxin derivatives, such as DcB with a known glaucoma drug Latanoprost, three groups of five rabbits were used. Rabbits treated with Latanoprost, received the medication every day for 5 days before the start of the experiment. On the day of the experiment rabbits were treated at 5 minutes interval with one drop of 1 mM DcB, one drop of 0.005% Latanoprost (Xalatan™, Pfizer) or one drop each of DcB and Latanoprost (RE), or normal saline (LE, Control). IOP was measured every hour for 12 hours (DcB and Latanoprost alone) and after 24 hours (DcB with Latanoprost). Basal IOPs in both eyes, without any medication, were measured 5 days before and on the day of the experiment. All eyes were examined routinely by ophthalmic examinations and were free of any abnormalities. Corneal thickness (μm) was measured using an ultrasonic pachometer (Sonogage pachometer, Cleveland, USA).

Stock solutions of the tested compounds were dissolved in ethanol, and freshly diluted in phosphate buffer (PBS) for each experiment, such that the final ethanol concentration did not exceed 1%.

The first set of experiments examined the effects of the α2-inhibitor compounds, according to embodiments of the present invention, when applied just before 4AP, used as a pharmacological tool to transiently raise IOP. One drop of the tested compound (0.01-0.3 mM) was applied topically to the rabbit's eyes prior to the 4AP, and IOP was then measured over 5 hours.

FIGS. 1A-D present comparative plots of IOP as a function of time, showing the dose response of α2-inhibitor compounds, according to some embodiments of the present invention, in lowering IOP in live rabbits, wherein FIG. 1A shows the results obtained for DiB, FIG. 1B shows the duration of the effect of DiB while 4AP is added every 2 hours so as to maintain the raised control IOP, FIG. 1C shows the results obtained for DMcP, and FIG. 1D shows the results obtained for DcB.

As can be seen in FIG. 1A, DiB, having Ki α1β1:α2β3 ratio of 16-fold) prevents the rise of the 4AP-induced IOP at concentrations of more than 0.030 mM while at 10 μM it is still effective. As can be seen in FIG. 1B, the duration of the DiB effect was shows that 1 mM DiB is effective for about 8 hours before the IOP begins to rise back up to control levels, which is a significantly long effect.

As can be seen in FIGS. 1B and 1C, DMcP, having a Ki α1β1:α2β3 ratio of 22-fold, and DcB, having a Ki α1β1:α2β3 ratio of 33-fold, exhibit similar IOP reduction as DiB (FIG. 1A), but in even lower concentrations. As can be seen in FIGS. 1B and 1C, low concentrations of 0.01 mM of DMcP or DcB are sufficient to prevent the 4AP-induced rise in IOP.

As can further be seen in FIGS. 1B and 1C, at higher concentrations of 0.1-0.3 mM, the IOP is reduced to levels which are significantly lower than the starting IOP. The latter observation implies that these compounds could reduce basal IOP even in the absence of 4AP.

FIGS. 2A-D present comparative plots of IOP as a function of time, demonstrating the capacity of the α2-inhibitor compounds, according to some embodiments of the present invention, to lower IOP below basal levels compared to a buffer control when administered topically to one eye of a rabbit, while the other eye received PBS as a control, wherein FIG. 2A shows the lack of effect of digoxin, FIG. 2B shows the lack of effect of DiB (a non-cyclic moiety inhibitor), FIG. 2C shows the notable of effect of DMcP, and FIG. 2D shows the notable of effect of DcB.

As can be seen in FIG. 2A-B, neither digoxin nor DiB had a significant effect on lowering the basal IOP. This observation coincides with other observations with digoxin or other non-cyclic moiety digoxin derivatives, DMe, DGlyN, the latter two exhibiting enhanced selectivity for α2β1.

As can be seen in FIGS. 2C-D, both DMcP and DcB significantly reduced the basal IOP by 20-25% (about 4 mm Hg for rabbit with a basal IOP of 17 mm Hg) over the test period of 4-5 hours. A higher concentration of DcB (2 mM) reduces the IOP similarly to 1 mM DcB.

This observation indicates that the α2-inhibitor compounds, according to some embodiments of the present invention, can be used effectively to treat medical conditions where there in a need to lower IOP below what is co considered to be a normal pressure, such as cases of low-tension glaucoma and normal-tension glaucoma.

A final set of experiments compared the effectiveness and duration of the effects of topical administration of DcB on basal IOP, with those of a widely used anti-glaucoma drug, Latanoprost, applied either alone or in combination (co-administration).

Groups of 5 rabbits were treated once a day for 5 days with Latanoprost and on the sixth day with DcB, Latanoprost, or DcB/Latanoprost combination. IOP measurements were made for the next 12 hours or over 24 hours for the group treated with both DcB and Latanoprost.

FIGS. 3A-C present comparative plots of IOP as a function of time, demonstrating the effect of α2-inhibitor compounds, according to some embodiments of the present invention, to potentiate the drug Latanoprost in lowering IOP below basal levels, wherein FIG. 3A shows the effect of DcB alone, FIG. 3B shows the effect of Latanoprost alone, and FIG. 3C shows the effect of co-administering DcB with Latanoprost.

As can be seen in FIGS. 3A-C, compared to the basal IOP values of 17-18 mmHg, after 3-8 hours the steady-state IOP was lower by 3.5±0.15, 2.6±0.11 and 3.44±0.39 mmHg with DcB, Latanoprost and DcB/Latanoprost, respectively, corresponding to steady-state IOP's of 75-80%, 85% and 75-80% of the unchanged control values.

As can further be seen in FIGS. 3A-C, DcB, an α2-inhibitor compound according to some embodiments of the present invention, was about 25% more effective than Latanoprost in reducing the basal (normal) levels of IOP.

The combination of DcB and Latanoprost was similar in effect to DcB alone; however, with respect to the duration of the effect, for either DcB and Latanoprost, applied alone, the IOP returned to the control (normal) value after 12 hours, but with the combined DcB/Latanoprost treatment the low IOP was maintained for a significantly longer period and it returned to the control value only after 24 hours.

Latanoprost was applied for 5 days prior to the day of measurement, due to reports that this pre-treatment produces optimal effects on IOP in humans, although in these experiments with rabbits it seems that this was unnecessary since the effects of Latanoprost were observed acutely and had dissipated completely after 24 hours.

The animals which received the treatment presented in FIGS. 3A-C were used for corneal thickness measurements after topically administering drops of DcB or Latanoprost to 4 rabbits once a day for 5 additional days, totaling 6 days of treatment with DcB or Latanoprost, and the results are presented in Table 4 below.

TABLE 4 Treatment to Pachymetry (mm) Rabbit RE RE LE 1 DcB 455 456 2 DcB 480 456 3 DcB 400 396 4 DcB 382 374 5 Latanoprost 467 471 6 Latanoprost 489 498

As seen in Table 4, there was no detectable effect of either drug on corneal thickness. In addition, by inspection, no significant redness or ocular irritation was observed.

In order to assess whether topical application of DcB damaged the tissues of the eye including cornea, iris, lens, cilary body, retina, choroid and sclera, a histological examination was conducted after topical application, using one drop of 1 mM DcB daily for one week in one eye of each animal, with the other eye treated topically with one drop of PBS and serving as the control. Animals were sacrificed, eyes were removed, fixed in 10% neutral buffered formalin, processed routinely for histological examination, trimmed at 4 μm, and stained with hematoxylin and eosin. No significant histological differences were observed in a comparative analysis of the treated and untreated eyes, indicating that topical treatment with DcB for a week does not cause tissue damage in the eye.

Example 6 Prodrug Synthesis and Characterization

Prodrugs of the compounds presented herein were developed in order to improve the pharmacokinetic profile of the active compounds, e.g., to avoid potential toxic effects in the cornea. The synthetic strategy included preparation of substantially inactive derivatives of the compounds, which are ineffective as an inhibitor of the major isoform of the Na,K-ATPase in the cornea (mainly the α1β1 isoform), and which can penetrate into the eye and thereafter are converted into the active form by biodegradation processes. The biodegraded and active form of the prodrug can then inhibits the α2β2/3 complex in the ciliary NPE cells and reduces inflow of aqueous humor.

Synthesis:

The synthesis of an exemplary prodrug compound, according to some embodiments of the present invention, 3′,3″-bisacetyl digoxin cyclobutane (bisAcDcB), from an exemplary compound according to some embodiments of the present invention, DcB, is illustrated in Scheme 2 below.

In this example, the bisacetyl derivative (IUPAC name (2R,3R,4S,6R)-3-(((2S,4S,5R,6R)-4-acetoxy-5-((4-cyclobutyl-2-methyl-1,4-oxazepan-7-yl)oxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-6-(((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2-methyltetrahydro-2H-pyran-4-yl acetate) was afforded by stirring for 6 hours at room temperature a solution of DcB (10 mg, 12.3 μmol, MW=817) and 4-dimethylaminopyridine (DMAP) catalyst (0.3 mg, 2.4 μmol, MW=122) in 2 ml toluene, 2 ml pyridine and 0.7 ml acetic anhydride.

Thereafter, the resulting mixture, rich in the bis-acetylated derivative, was extracted with water and dichloromethane, and subjected to final wash with HCl-acidified water pH of about 4. The resulting residue was dried over MgSO₄, and purified in normal phase HPLC using a silica gel column.

The synthesis of an exemplary prodrug compound, according to some embodiments of the present invention, 12,3′,3″-trisacetyl digoxin cyclobutane (trisAcDcB), from an exemplary compound according to some embodiments of the present invention, DcB, is illustrated in Scheme 3 below.

In this example, the trisacetyl derivative (IUPAC name (2R,3R,4S,6R)-6-(((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12-acetoxy-14-hydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-(((2S,4S,5R,6R)-4-acetoxy-5-((4-cyclobutyl-2-methyl-1,4-oxazepan-7-yl)oxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-methyltetrahydro-2H-pyran-4-yl acetate) was afforded by stirring for 24 hours at room temperature a solution of DcB (10 mg, 12.3 μmol, MW=817) and 4-dimethylaminopyridine (DMAP) catalyst (0.3 mg, 2.4 μmol, MW=122) in 2 ml toluene, 2 ml pyridine and 0.7 ml acetic anhydride.

Thereafter, the resulting mixture, rich in the tris-acetylated derivative, was extracted with water and dichloromethane, and subjected to final wash with HCl-acidified water pH of about 4. The resulting residue was dried over MgSO₄, and purified in normal phase HPLC using a silica gel column.

Calculated octanol-water partition coefficient (C log P) for DcB is 3.43, for a mono-acetyl derivative of DcB is 3.93, for the bisAcDcB derivative is 4.37, and for the trisAcDcB derivative is 4.85 (C Log P software ALOGPS 2.1 [Tetko, I. V. et al., J. Chem. Inf. Comput. Sci., 2002, 42, 1136-45])

In Vitro Inhibition Activity:

The results of the inhibition assay of purified human Na,K-ATPase (α1β1FXYD1 and α2β1FXYD1) by DcB, bisAcDcB and trisAcDcB are presented as Ki values in Table 5 below.

TABLE 5 Ki of α1β1FXYD1 Ki of α2β1FXYD1 Compound [nM] [nM] DcB 135 ± 11    8 ± 1.25 BisAcDcB 1180 ± 280 395 ± 50 TrisAcDcB 13200 ± 6500 2500 ± 490

As can be seen in Table 5, the Ki values of α1β1 by the exemplary bisAcDcB prodrug derivative and the exemplary trisAcDcB prodrug derivative are about 9-fold and 100-fold higher than that observed for the corresponding exemplary compound DcB, respectively, while the Ki values of α2β1 by bisAcDcB and trisAcDcB are about 50-fold and 300-fold higher than that observed for the corresponding DcB, respectively.

In Vivo Activity—Lowering of Basal IOP:

One 30 μl drop of trisAcDcB or vehicle was applied to the right or left eye of two rabbits, respectively, at 0.05, 0.1, and 0.2 mM concentrations. The IOP was then measured every 1-2 hours over 12 hours.

FIG. 4 presents comparative plots of IOP as a function of time, demonstrating the capacity of the trisAcDcB prodrug of the α2-inhibitor compound DcB, according to some embodiments of the present invention, to lower IOP below basal levels compared to a buffer control when administered topically to one eye of a rabbit, while the other eye received PBS as a control.

As can be seen in FIG. 4, no effect was observed at the first four hours, and after that the IOP in the drug-treated samples dropped from 15-16 mmHg to a minimum of 12 mmHg within 5-6 hours, before returning to the control level after 12 hours. In addition, the maximal effect of the trisAcDcB on IOP was essentially equal to that produced by the parent DcB (see, for example, FIG. 2D). The comparison with the parent DcB compound shows that significantly lower doses of the prodrug are required to produce the maximal effect.

Thus, it can be concluded that acetyl prodrugs of the compounds presented herein exhibit low affinity and weak inhibition of α1β1 isoform, which is the major isoform in the cornea, making it unlikely that topical application of the prodrug in human eye would cause corneal swelling or other toxic adverse effects. The lag of four hours prior to a detectable effect of the prodrug suggests that this time is required for the intra-ocular esterases to hydrolyze the acetyl groups and regenerate DcB within the anterior chamber of the eye. Moreover, significantly lower doses of prodrug are required to achieve a comparable effect of the parent compound. This finding is consistent with a higher permeability through the cornea compared to DcB (C log P 3.43) due to a higher lipophilicity of the esters (triAcDcB C log P 4.85).

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety. 

What is claimed is:
 1. A pharmaceutical composition comprising as active ingredients: at least one ingredient selected from the group consisting of a prostaglandin analog, a β-blocker, an adrenergic agent, an α2-adrenergic receptor agonist, a miotic agent, a carbonic anhydrase inhibitor and a cholinergic agonist; and a compound represented by Formula III:

including any pharmaceutically acceptable salt, hydrate, solvate, enantiomer and diastereomer thereof, and any mixtures thereof, and a pharmaceutically acceptable carrier, wherein: X is H or OH; R′ is selected from the group consisting of OH, C₁-C₆ alkyl, C₁-C₆ haloalkyl (C₁-C₆ alkyl substituted with at least one halo), —(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂, —(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂ and —(CR^(b)R^(c))n-NH₂; Y is O or S; R1, R2 and R3 are each independently H or a C₁-C₄ alkyl; Ra is a C₁-C₄ alkyl; Rb, Rc and Rd are each independently selected from H, a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; Re is selected from a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; and n is 0, 1 or
 2. 2. The pharmaceutical composition of claim 1, being packaged in a packaging material and identified in print, or on said packaging material, for use in reducing intraocular pressure (IOP).
 3. The pharmaceutical composition of claim 1, wherein said at least one ingredient is said prostaglandin analog.
 4. The pharmaceutical composition of claim 1, wherein said at least one ingredient is said β-blocker.
 5. The pharmaceutical composition of claim 1, wherein said at least one ingredient is said adrenergic agent.
 6. The pharmaceutical composition of claim 1, wherein said at least one ingredient is said α2-adrenergic receptor agonist.
 7. The pharmaceutical composition of claim 1, wherein said at least one ingredient is said miotic agent.
 8. The pharmaceutical composition of claim 1, wherein said at least one ingredient is said carbonic anhydrase inhibitor.
 9. The pharmaceutical composition of claim 1, wherein said at least one ingredient is said cholinergic agonist.
 10. A method of treating a heart condition in a subject in need thereof, comprising co-administering to the subject a therapeutically effective amount of: an agent selected from the group consisting of a β-blocker, an anticoagulation agent, an angiotensin-converting-enzyme inhibitor and an angiotensin II receptor antagonist; and a compound represented by Formula III:

including any pharmaceutically acceptable salt, hydrate, solvate, enantiomer and diastereomer thereof, and any mixtures thereof, wherein: X is H or OH; R′ is selected from the group consisting of OH, C₁-C₆ alkyl, C₁-C₆ haloalkyl (C₁-C₆ alkyl substituted with at least one halo), —(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂, —(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂ and —(CR^(b)R^(c))n—NH₂; Y is O or S; R₁, R₂ and R₃ are each independently H or a C₁-C₄ alkyl; Ra is a C₁-C₄ alkyl; Rb, Rc and Rd are each independently selected from H, a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; Re is selected from a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; and n is 0, 1 or
 2. 11. The method of claim 10, wherein said agent is said β-blocker.
 12. The method of claim 10, wherein said agent is said anticoagulation agent.
 13. The method of claim 10, wherein said agent is said angiotensin-converting-enzyme inhibitor.
 14. The method of claim 10, wherein said agent is said angiotensin II receptor antagonist. 